FAQ - ROJE Technologies

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Nucleic Acid Isolation

DNA Technologies

DNall Kit

Low DNA yield

1. Inappropriate sample storage condition
-Please refer to sample preparation guidelines.

2. Insufficient lysis
– Too much starting material results in low DNA yield
– Make sure to do pulse-vortexing vigorously after addition of lysis buffer and RJ-Protease.
– Incubate mixture of the sample and lysis buffer for an additional 15-20 min at 56° C.
– Ensure mixing sample completely before incubation step.

3. Too few cells in the sample
– Do the test with new samples

4. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

5. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

6. DNA elution is incomplete
– Perform rehydration step once more, by adding another 50-200µl rehydration buffer to column and incubate at room temperature before centrifugation.
– Check that all previous steps are done appropriately.

7. DNA improperly eluted
– The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

Degradation

1. Sample contaminated with DNase
– Be sure to do the process in accordance with the reference protocol.

2. The genomic DNA was handled improperly
– Reduce vertexing times during mixing steps (not more than recommended).

3. Improper sample storage
– Please refer to sample preparation guidelines.

4. Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

Low 260/280 ratio

1. Sample was diluted in water
-It is recommended to use ROJE rehydration buffer for DNA elution, however if you want to use water instead make sure that the pH is at least 7 or use 10 mM Tris-HCl Ph≥ 7.0.

2. Protein contamination
– This is often due to exceeding the amount of starting material. Follow precisely the respective protocol. If DNA purification is still problematic further reduce the amount of starting material.

High 260/280 ratio

1. RNA contamination
– This kit is optimized to extract DNA without RNA contamination. However, if you need to make sure that no
RNA contamination is present, you can purchase Prime-RNase A (Cat No. EB983013) separately and perform RNase treatment during the process.

DNA does not perform well in downstream applications

1. PCR reaction condition is not optimized
-Make sure that PCR condition is optimized by testing:
. Primer designs and annealing conditions
. Changing source of Taq Polymerase
. Different amount of DNA sample

2.DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

3. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

4. Do not use standard buffer for DNA rehydration
– Use ROJE Rehydration buffer for dissolving purified DNA.

Clogged Column

1. Maximum amount of sample exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.
2. The lysate mixture is not homogeneous
– To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.
3. The sample is too much
– Use less starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column.

This post is also available in:
Persian

DNall Plus kit

Low DNA yield

1. Inappropriate sample storage condition
– Please refer to sample preparation guidelines.

2. Insufficient lysis
-Too much starting material results in low DNA yield. To optimize the results, refer to Table 2.
– Make sure to do pulse-vortexing vigorously after addition of lysis buffer and RJ-Protease.
– Incubate mixture of the sample and lysis buffer for an additional 15-20 min at 56° C.
– Ensure mixing sample completely before incubation step.

3.Too few cells in the sample
– Do the test with new samples.

4. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

5. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

Degradation

1. Sample contaminated with DNase
-Be sure to do the process in accordance with the reference protocol.

2. The genomic DNA was handled improperly
– Reduce vertexing times durin mixing steps (not more than recommended).

3. Improper sample storage
– Please refer to sample preparation guidelines.

4. Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

Low 260/280 ratio

1. Sample was diluted in water
– It is recommended to use ROJE rehydration buffer for DNA elution, however if you want to use water instead make sure that the pH is at least 7 or use 10 mM Tris-HCl Ph≥ 7.0.

High 260/280 ratio

1. RNA contamination
– This kit is optimized to extract DNA without RNA contamination. However, if you need to make sure that no RNA contamination is present, you can purchase PrimeRNase A (Cat No. EB983013) separately and perform RNase treatment during the process.

DNA does not perform well in downstream applications

1. PCR reaction condition is not optimized
Make sure that PCR condition is optimized by testing:
– Primer designs and annealing conditions
– Changing source of Taq Polymerase
– Different amount of DNA sample

2. DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

3. Ethanol carryover
-Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

4. Do not use standard buffer for DNA rehydration
– Use ROJE Rehydration buffer for dissolving purified DNA.

Clogged Column

1. Maximum amount of sample exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

2. The lysate mixture is not homogeneous
– To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.

3. The sample is too much
– Use less starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column.

This post is also available in:
Persian

DNall VirAll Kit

Low DNA yield

1. Insufficient lysis
– Please refer to Table 2 to apply best match for size of starting material and amount of lysis buffer.
– Make sure to do pulse-vortexing after addition of lysis buffer and RJ-Protease.

2. Too few virus in the sample
– Do the test with new samples.

3. Incomplete lysing
-Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

4. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

5. DNA improperly eluted
– The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

DNA does not perform well in downstream applications

1. DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

Low RNA Yield

1. Carrier RNA not added to GLB
– Reconstitute carrier RNA in ERR and mix with GLB as described before. Repeat the purification process with new samples.

2. Carrier RNA is degraded
– After reconstitution in ERR, not stored at -15̊ C to -30 ̊C.
– Multiple freeze–thaw cycles. In each cases, reconstitute RNA carrier in ERR again and prepared new GLB. Then, repeat the procedure.

3. Multiple freeze–thaw cycles on sample
– Do not freeze and thaw sample more than once

4. Forget to add ethanol to lysate
– Repeat the procedure with new sample.

5. Low percentage ethanol used
– Repeat the procedure with new sample. Use 96–100% ethanol.

6. RNA degraded
– It may happen that RNA is degraded by RNases in the starting material (plasma, serum, body fluids). Use Nuclease-free water and make sure no RNase is presented during the procedure.

7. BWB1 or BWB2 prepared incorrectly
-Referred to some tips to know, check the BWB1 and BWB2 dilution process and repeat the procedure again.

8. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

RNA does not perform well in downstream applications

1. Too much carrier RNA in the eluate
– Determine the maximum amount of carrier RNA suitable for your RT-PCR. Adjust the concentration of carrier RNA added to GLB.

2. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

DNA contamination

1. Co-purification of genomic DNA
– To avoid co-purification of genomic DNA, use of cell-free body fluids. Samples containing cells, such as cerebrospinal fluid, bone marrow, urine and most swabs, should be made cell-free by centrifugation or filtration. If using centrifugation, pellet the cells for 10 min at 1500 x g and use supernatant for isolation of viral RNA. If DNA-free RNA is required, digest either the sample or the eluate with RNase-free DNase. DNase in the eluate must be inactivated by heat treatment (15 min, 70°C)

Column clogging

1. Precipitates were not removed.
– When using plasma samples, remove visible Cryoprecipitates by centrifugation for 5 min at 3000 × g

2. Lysate not completely passed through the membrane
– Centrifuge for 1 min at full speed or until all the lysate has passed through the membrane.

This post is also available in:
Persian

DNJia Amnio Pure Kit

Low DNA yield

2. Too few amniocytes in the sample
– Do the test with new samples.

3. Incomplete lysing
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

4. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

Poor DNA Quality

1. RNA can be copurified with the genomic DNA
– RNase treatment can be performed.

2. Incomplete cell lysis
– Incubate sample with lysis buffer and RJ-Protease for an extra 5- 10 minutes.

DNA does not perform well in downstream applications

1. DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

This post is also available in:
Persian

DNJia Blood & Cell Kit

Low DNA yield

1. The blood sample was too old
– Please refer to sample preparation guidelines.

2. Insufficient lysis
– Please refer to Table 2 to apply best match for size of starting material and amount of lysis buffer.
– Make sure to do pulse-vortexing vigorously after addition of lysis buffer and RJ-Protease.
– Incubate mixture of the sample and lysis buffer for an additional 15-20 min at 56° C.

3. Too few white blood cells in the sample
– Do the test with new blood samples.

4. Whole blood sample was not mixed before processing
– White blood cells should be in suspension. So, make sure to mix whole blood samples before processing.

5. Incomplete lysing of WBC’s
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

6. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

7. DNA improperly eluted
– The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you
want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

Degradation

1. Sample contaminated with DNase
– Be sure to do the process in accordance with the reference protocol.

2. Inappropriate sample collection or storage of starting material
– Please refer to sample preparation guidelines.

3.Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

Poor DNA Quality

1. RNA can be copurified with the genomic DNA
– RNase treatment can be performed.

2. Incomplete cell lysis
– Incubate sample with lysis buffer and RJ-Protease for an extra 5- 10 minutes.

DNA does not perform well in downstream applications

1. DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

This post is also available in:
Persian

DNJia Plus Blood & Cell Kit

Low DNA yield

1. The blood sample was too old
– Please refer to sample preparation guidelines.

3. Too few white blood cells in the sample
– Do the test with new blood samples.

4. Whole blood sample was not mixed before processing
– White blood cells should be in suspension. So, make sure to mix whole blood samples before processing.

5. Incomplete lysing of WBC’s
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

6. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

7. DNA improperly eluted
– The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you
want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

Degradation

1. Sample contaminated with DNase
– Be sure to do the process in accordance with the reference protocol.

2. Inappropriate sample collection or storage of starting material
– Please refer to sample preparation guidelines.

3. Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

Poor DNA Quality

1. RNA can be copurified with the genomic DNA
– RNase treatment can be performed.

2. Incomplete cell lysis
– Incubate sample with lysis buffer and RJ-Protease for an extra 5- 10 minutes.

DNA does not perform well in downstream applications

1. DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

This post is also available in:
Persian

DNJia Plus Tissue and Bacteria Kit

Low yield

1. Inappropriate sample storage condition
– Best storage condition for tissue is at – 20°C or -70°C. Avoid freezing and thawing of samples, which results in reducing size of DNA.
2. Incomplete cell lysis
– Too much starting material results in low DNA yield. to optimize the results, refer to Table 2.

3. Insufficient mixing of Sample with TLB
– Ensure mixing sample completely before incubation step.

4. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure n remaining of Ethanol on column.

5. DNA elution is incomplete
– Perform rehydration step once more, by adding another 50-200µl rehydration buffer to column and incubate at room temperature before centrifugation.
– Check that all previous steps are done appropriately.

6. DNA was eluted with water instead of Elution Buffer
– The best buffer for DNA rehydration is prepared in the Kit Box. We insist to use the supplied rehydration buffer, however if you want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

Degradation

1. Sample is too old
– Old samples may have degraded DNA in the rehydration step.

2. Improper sample storage
– Refer to sample preparation tips.

3. The genomic DNA was handled improperly
– Reduce vertexing times during mixing steps (not more than recommended)

Low 260/280 ratio

1. Insufficient lysis
-Increase incubate time in lysis buffer

2. Sample was diluted in water
– It is recommended to use ROJE rehydration buffer for DNA elution, however if you want to use water instead make sure that the pH is at least 7 or use 10 mM Tris-HCl Ph≥ 7.0.

3. Protein contamination
– This is often due to exceeding the amount of starting material. Follow precisely the respective protocol, If DNA purification is still problematic further reduce the amount of starting material.

High 260/280 ratio

1. RNA contamination
– This kit is optimized to extract DNA without RNA contamination. However, if you need to make sure that no RNA contamination is present, you can purchase Prime-RNase A (Cat No. EB983013) separately and perform RNase treatment during the process or on the prepared DNA.

Not performing well in downstream application

1. PCR reaction condition is not optimized
– Make sure that PCR condition is optimized by testing:
– Primer designs and annealing conditions
– Changing source of Taq Polymerase
– Different amount of DNA sample

2. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

3. Do not use standard buffer for DNA rehydration
– Use ROJE Rehydration buffer for dissolving purified DNA.

Clogged Column

1. Maximum amount of tissue exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

2. The lysate mixture is not homogeneous
– To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.

3. The sample is too large
– Use less starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column

This post is also available in:
Persian

DNJia Tissue and Bacteria Kit

Low yield

1. Inappropriate sample storage condition
– Best storage condition for tissue is at – 20°C or -70°C. Avoid freezing and thawing of samples, which results in reducing size of DNA.
2. Incomplete cell lysis
-Too much starting material results in low DNA yield. to optimize the results, refer to Table 2.

3. Insufficient mixing of Sample with TLB
– Ensure mixing sample completely before incubation step.

4. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

Degradation

1. Sample is too old
– Old samples may have degraded DNA in the rehydration step.

2. Improper sample storage
– Refer to sample preparation tips.

3. The genomic DNA was handled improperly
– Reduce vertexing times during mixing steps (not more than recommended)

Low 260/280 ratio

1. Insufficient lysis
– Increase incubate time in lysis buffer

High 260/280 ratio

1. RNA contamination
– This kit is optimized to extract DNA without RNA contamination. However, if you need to make sure that no RNA contamination is present, you can purchase Prime-RNase A (Cat No. EB983013) separately and perform RNase treatment during the process or on the prepared DNA.

Not performing well in downstream application

2. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

3. Do not use standard buffer for DNA rehydration
– Use ROJE Rehydration buffer for dissolving purified DNA.

Clogged Column

1. Maximum amount of tissue exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

2. The lysate mixture is not homogeneous
– To ensure a homogeneous solution, vortex for 10-15 seconds before applying the lysate to the spin column.

3.The sample is too large
– Use less starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column

This post is also available in:
Persian

DNJia VirAll Kit

Low DNA yield

2. Too few virus in the sample
– Do the test with new samples.

3. Incomplete lysing
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

4.Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

DNA does not perform well in downstream applications

1. DNA was not washed with the provided washing buffer
-Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

Low RNA Yield

1. Carrier RNA not added to GLB
– Reconstitute carrier RNA in ERR and mix with GLB as described before. Repeat the purification process with new samples.

3. Multiple freeze–thaw cycles on sample
-Do not freeze and thaw sample more than once

4. Forget to add ethanol to lysate
– Repeat the procedure with new sample.

5. Low percentage ethanol used
– Repeat the procedure with new sample. Use 96–100% ethanol.

7. WB1 or BWB2 prepared incorrectly
-Referred to some tips to know, check the BWB1 and BWB2 dilution process and repeat the procedure again.

8. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

RNA does not perform well in downstream applications

1. Too much carrier RNA in the eluate
– Determine the maximum amount of carrier RNA suitable for your RT-PCR. Adjust the concentration of carrier RNA added to GLB.

2. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

DNA contamination

– If DNA-free RNA is required, digest either the sample or the eluate with RNase-free DNase. DNase in the eluate must be inactivated by heat treatment (15 min, 70°C)

Column clogging

1. Precipitates were not removed.
– When using plasma samples, remove visible Cryoprecipitates by centrifugation for 5 min at 3000 × g

2. Lysate not completely passed through the membrane
– Centrifuge for 1 min at full speed or until all the lysate has passed through the membrane.

This post is also available in:
Persian

DNJia Virus DNA Kit

Low DNA yield

2. Too few virus in the sample
– Do the test with new samples.

3. Incomplete lysing
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

4.Reagents not applied correctly
– Prepare buffers according to the protocol.
– Make sure Ethanol is added to BWB1 and BWB2.
– Repeat the procedure with a new sample.

Poor DNA Quality

1. RNA can be copurified with the viral DNA
-RNase treatment can be performed.

2.Incomplete cell lysis
– Incubate sample with lysis buffer and RJ-Protease for an extra 5-10 minutes.

Column clogging

1. Starting material was not completely disrupted
– Reduce the amount of starting material and increase disruption time.

2. Precipitates were not removed.
– When using plasma samples, remove visible Cryoprecipitates by centrifugation for 5 min at 3000 × g

DNA does not perform well in downstream applications

1.DNA was not washed with the provided washing buffer
– Ensure the column was washed once with prepared BWB1 and once more with prepared BWB2, respectively.

2.Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

This post is also available in:
Persian

DNSol Clotted Blood Kit

Low DNA yield

1. Insufficient lysis
– Not forget to add appropriate lysis buffer in accordance with the reference protocol.
– Make sure to do pulse-vortexing vigorously after addition of lysis buffer.

2. Too few white blood cells in the sample
– Do the test with new samples.

3. Incomplete lysing of WBC’s
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

4. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Repeat the procedure with a new sample.

5. DNA improperly eluted
– The best buffer for DNA re-hydration is prepared in the Kit Box. We insist to use the supplied re-hydration buffer, however if you
want to use water instead, make sure that the pH is at least 7.0, or use 10 mM Tris-HCl Ph≥ 7.0.

Degradation

1. Sample contaminated with DNase
– Be sure to do the process in accordance with the reference protocol.

2. Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

DNA does not perform well in downstream applications

1. Ethanol carryover
– Ensure that the traces of Ethanol before re-hydration step is removed.

This post is also available in:
Persian

DNSol Kit

Low DNA yield

1. The blood sample was too old
– Please refer to sample preparation guidelines.

2. Insufficient lysis
– Not forget to add appropriate lysis buffer in accordance with the reference protocol.
– Make sure to do pulse-vortexing vigorously after addition of lysis buffer.

3. Too few white blood cells in the sample
– Do the test with new blood samples.

4. Whole blood sample was not mixed before processing
– White blood cells should be in suspension. So, make sure to mix whole blood samples before processing.

5. Incomplete lysing of WBC’s
– Repeat the reaction once more and make sure to mix the sample and lysis buffer completely by pulse-vortexing.

6. Reagents not applied correctly
– Prepare buffers according to the protocol.
– Repeat the procedure with a new sample.

Degradation

1. Sample contaminated with DNase
– Be sure to do the process in accordance with the reference protocol.

2. Inappropriate sample collection or storage of starting material
– Please refer to sample preparation guidelines.

3. Too old sample
– Old samples stored at inappropriate conditions always yield sheared DNA.

Poor DNA Quality

1. RNA can be copurified with the genomic DNA
– RNase treatment can be performed.

DNA does not perform well in downstream applications
1. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed.

This post is also available in:
Persian

Gel and PCR Purification

Gel & PCR Purification Set

Low nucleic acid recovery

1. Incomplete solubilizing of gel slice
– It may be due to, large sample or inappropriate RGB amount. Repeat procedure, with smaller starting material and appropriate RGB amount.

2. Too large gel slice
– The most recovery can be obtained from less than 200 mg gel. However, for size bigger than 200 mg, it is recommended to use multiple spin column.

3. RGB turned yellow or orange
– RGB color should be pink, however if it changes to yellow or orange, set the pH at 5.00 by using H2SO4.

4. GWB did not contain Ethanol
– Please sure to add appropriate Ethanol to GWB, before first use.

5. Absence of PCR amplification
– Run the PCR product on gel before and after purification to make sure that the PCR amplification was done properly.

6. Using not prepared RPB
– Make sure to add Absolute Ethanol to RPB (30% of total volume), before first use.

DNA does not perform well in downstream application

1. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

2. Salt concentration in elution
– After adding 750 µl of GWB, incubate 5 min at room temperature, then centrifuge it.

3. Presence of Primer-primer dimer in DNA elution
– To completely remove primerprimer dimers, perform one additional step before adding GWB. Add 750 µl of a 35-40% guanidine hydrochloride aqueous solution (35 g in 100 ml). Then continue the procedure by performing GWB to spin column.

4. Presence of ssDNA, appears as smear band on a gel electrophoresis
Select one of these ways to reanneal the ssDNA:
– Incubate the mixture at 95 ̊C for 3 min then allow them to cool slowly at room temperature.
– Elute the DNA in 10 mM Tris buffer containing 10 mM NaCl.

Note: If using second method, consider, salt concentration for downstream application.

This post is also available in:
Persian

Geljia Kit

Low nucleic acid recovery
1. Incomplete solubilizing of gel slice
– It may be due to, large sample or inappropriate RGB amount. Repeat procedure, with smaller starting material and appropriate RGB amount.

2. Too large gel slice
– The most recovery can be obtained from less than 200 mg gel. However, for size bigger than 200 mg, it is recommended to use multiple spin column.

3. RGB turned yellow or orange
– RGB color should be pink, however if it changes to yellow or orange, set the pH at 5.00 by using H2SO4.

4. GWB did not contain Ethanol
– Please sure to add appropriate Ethanol to GWB, before first use.

DNA does not perform well in downstream application

1. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

2. Salt concentration in elution
– After adding 750 µl of GWB, incubate 5 min at room temperature, then centrifuge it.

This post is also available in:
Persian

PCR-Pure Kit

Low nucleic acid recovery

1. Using not prepared RPB
– Make sure to add Absolute Ethanol to RPB (30% of total volume), before first use.

2. GWB did not contain Ethanol
– Not forget to add appropriate amount of Ethanol to GWB, before first use.

3. Absence of PCR amplification
– Run the PCR product on gel before and after purification to make sure that the PCR amplification was done properly.

DNA does not perform well in downstream application

1. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

2. Salt concentration in elution
– After adding 750 µl of GWB, incubate 5 min at room temperature, then centrifuge it.

3. Presence of Primerprimer dimer in DNA elution
– To completely remove primer-primer dimers, perform one additional step before adding GWB. Add 750 µl of a 35-40% guanidine hydrochloride aqueous solution (35 g in 100 ml). Then continue the procedure by performing GWB to spin column.

Note: If using second method, consider, salt concentration for downstream application.

This post is also available in:
Persian

Safe PCR-Pure Kit

Low nucleic acid recovery

1. GWB did not contain Ethanol
– Not forget to add appropriate amount of Ethanol to GWB, before first use.

2. Absence of PCR amplification
– Run the PCR product on gel before and after purification to make sure that the PCR amplification was done properly.

DNA does not perform well in downstream application

1. Ethanol carryover
– Ensure that the traces of Ethanol before rehydration step is removed

2. Salt concentration in elution
– After adding 450 µl of GWB, incubate 5 min at room temperature, then centrifuge it.

3. Presence of ssDNA, appears as smear band on a gel electrophoresis
Select one of these ways to reanneal the ssDNA:
– Incubate the mixture at 95 ̊C for 3 min then allow them to cool slowly at room temperature.
– Elute the DNA in 10 mM Tris buffer containing 10 mM NaCl.

Note: If using second method, consider, salt concentration for downstream application.

This post is also available in:
Persian

RNA Technologies

RNaseLag

RNA Degradation

1. Stabilized old tissue
– Use fresh tissue. Immediately after harvesting sample, submerge sample in RNaseLag.

2. Not appropriate amount of RNaseLag
– Weight your sample before starting the procedure and use 10 µl RNaseLag per 1mg of tissue.

3.Too thick sample for stabilization
– Cut samples into slices less than 5 mm and then submerge in RNaseLag.

4. RNase contamination during RNA purification
– RNases can be introduced during RNA isolation. Refer to Appendix 1 for more information.

5. Storage duration is exceeded in RNaseLag
• Refer to Table 1.

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RNJia Lipid Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.
– For better results, it is recommended to store samples in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining trace of Ethanol on column.
– carefully remove the column from the collection tube so that the column does not contact the flow-through.

4. RNA elution is incomplete
– Perform rehydration step once more, by adding another 30-100 µl rehydration buffer to the column and before centrifugation, incubate 5 min at Room Temperature.
– Check that all previous steps are done appropriately.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag, Refer to page 9.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, based on sample type refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease free water with pH ≥ 7.5.

3. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase Treatment.
– RNSol pH might alter during storage. Check the RNSol pH it should be around

4. Protein contamination
– This is often due to exceeding the amount of starting material. Follow precisely the respective protocol, If RNA purification is still problematic further reduce the amount of starting material.
– Remove the aqueous phase precisely.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

3. Check RNSol pH
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeds kit specifications
– Refer to specifications to determine if the amount of starting material falls within kit specifications.

2. The sample is too large
– Use fewer starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column

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RNjia Bacteria Kit

Low yield

1. Incorrect starting material
– Refer to appendix 7 for information about cell counting and Table 2 for correct amount of starting material.

2. Incomplete cell wall distraction
– While using lysozyme, it may be needed to optimize lysozyme concentration and digestion time.
– While using mechanical disruption, it may be needed to lengthen the disruption duration.
– Freezing and thawing of the cell pellets treated by RNaseLag makes cell walls disruption easier.

3. Not harvesting cells during logarithmic phase
– For highest RNA yield, it is recommended to harvest cells at logarithmic growth phase.

4. Ethanol from the washing buffer is present at elution
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.
– Carefully remove the column from the collection tube so that the column does not contact the flow-through.

5. RNA elution is incomplete
– Perform rehydration step once more, by adding another 30 µl rehydration buffer to column and before centrifugation, incubate 5 min at 60 ̊C.
– Check that all previous steps are done appropriately.

Degradation

1. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

2. DNA contamination
– Follow precisely the respective protocol, if RNA purification is still problematic further do DNase Treatment.

3. Protein contamination
– This is often due to exceeding the amount of starting material. Follow precisely the respective protocol; if RNA purification is still problematic further reduce the amount of starting material.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at room temperature after addition of TWB1 and before centrifuging

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of starting material exceeds the kit specifications
– Refer to specifications to determine if amount of starting material falls within the kit specifications.

2. The sample is too large
– Use fewer starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period until the lysate passes through the column.

3. Centrifuge at low temperature
– The centrifugation temperature should be 20–25°C. Make sure that the centrifuge temperature is set at 25°C.

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RNjia Fibrous Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reducing RNA Yield.
– For better results, it is recommended to store sample in RNaseLag.

2. Incomplete cell lysis
– Too much starting material is resulting in low RNA yield. To optimize the results, refer to Table 2.

3. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, refer to sample preparation.

4. Ethanol from the washing buffer is present at elution
– Preform another centrifugation before rehydration step to ensure no remaining of ethanol on column.
– Carefully remove the column from the collection tube so that the column does not contact the flow-through.

5. RNA elution is incomplete
– Perform rehydration step once more, by adding another 30 µl rehydration buffer to column and centrifuge at 12000 for 1 min.
– Check that all previous steps are done appropriately.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store sample at RNaseLag.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, refer to sample preparation.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

3. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase
Treatment.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeds kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

3. Centrifuge at low temperature
– The centrifugation temperature should be 20–25°C. be sure that the centrifuge temperature is set at 25°C.

4. Centrifugation before adding ethanol is not performed (for animal tissue)
– Centrifuge the lyset before adding ethanol, and use supernatant for next step.

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RNJia Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reducing RNA Yield.
– For better results, it is recommended to store sample in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Insufficient disruption and homogenization
– If working with fibrous tissue, it is recommended to use RNJia Fibrous kit (Order by Cat No RN983024, RN983025 and RN983026)
– If working with bacteria, it is recommended to use RNJia Bacteria Kit (Order by Cat No RN983020, RN983021, RN983022 and RN983023)
– As a guide for better disruption and homogenization, based on sample type refer to suitable sample preparation guidelines.

5. RNA rehydration is incomplete
– Perform rehydration step once more, by adding another 30-50 µl nucleasefree water to column and before centrifugation, incubate 5 min at room temperature.
– Check that all previous steps are done appropriately.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store sample in RNaseLag.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– If working with fibrous tissue, it is recommended to use RNJia Fibrous Kit (Order by Cat No RN983024, RN983025 and RN983026)
– If working with bacteria, it is recommended to use RNJia Bacteria Kit (Order by Cat No RN983020, RN983021, RN983022 and RN983023)
– As a guide for better disruption and homogenization, based on sample type refer to suitable sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

3. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase Treatment.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at room temperature after addition of TWB1 and before centrifuging.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

3. Centrifuge at low temperature
– The centrifugation temperature should be 20–25°C. Make sure that the centrifuge temperature is set at 25°C.

4. Centrifugation before adding Ethanol is not performed (for animal tissue)
– Centrifuge the lysate before adding Ethanol, and use supernatant for next step.

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RNJia PB Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.
– For better results, it is recommended to store samples in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining trace of Ethanol on column.
– Carefully remove the column from the collection tube so that the column does not contact the flow-through.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag

2. Improper sample storage
– It is suggested to store samples in RNaseLag, Refer to page 9.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

3. Check RNSol pH
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of cells exceeds kit specifications
– Refer to specifications to determine if the amount of starting material falls within kit specifications.

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RNJia Phenol-free PB Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Ethanol from the washing buffer is present in elution
– Preform another centrifugation before rehydration step to ensure no remaining trace of Ethanol on column.
– Carefully remove the column from the collection tube so that the column does not contact the flow-through.

4. RNA elution is incomplete
– Perform rehydration step once more, by adding another 30-50 µl rehydration buffer to the column and before centrifugation, incubate 5 min at Room Temperature.
– Check that all previous steps are done appropriately.

Degradation

1. RNase contamination
– All buffers have been tested and are guaranteed RNasefree. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

3. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase Treatment.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flowthrough from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum number of cells exceeded kit specifications
– Refer to specifications to determine if amount of starting material falls within kit specifications.

2. The sample is too much
– Use fewer starting material. The problem can be solved by increasing the g-force and/or centrifuging for a longer period of time until the lysate passes through the column.

3. Centrifuge at low temperature
– The centrifugation temperature should be 20– 25°C. Make sure that the centrifuge temperature is set at 25°C.

4. Centrifugation before adding Ethanol is not performed (for animal tissue)
– Centrifuge the lysate before adding Ethanol, and use supernatant for next step.

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RNJia Virus Kit

Low RNA yield

1. Carrier RNA not added to BFC
– Reconstitute carrier RNA in ERR and mix with BFC as described before. Repeat the purification process with new samples.

3. Multiple freeze–thaw cycles on sample
-Do not freeze and thaw sample more than once

4. Forget to add ethanol to lysate
– Repeat the procedure with new sample.

5. Low percentage ethanol used
– Repeat the procedure with new sample. Use 96–100% ethanol.

7. BWB1 or BWB2 prepared incorrectly
– Referred to some tips to know, check the BWB1 and BWB2 dilution process and repeat the procedure again.

8. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

RNA does not perform well in downstream applications

1. Too much carrier RNA in the eluate
– Determine the maximum amount of carrier RNA suitable for your RT-PCR. Adjust the concentration of carrier RNA added to BFC.

2. BWB1 and BWB2 used in the wrong order
– Make sure to use BWB1 and BWB2 in write order.

DNA contamination

1. Co-purification of genomic DNA
– To avoid co-purification of genomic DNA, use of cell-free body fluids. Samples containing cells, such as cerebrospinal fluid, bone marrow, urine and most swabs, should be made cell-free by centrifugation or filtration. If using centrifugation, pellet the cells for 10 min at 1500 x g and use supernatant for isolation of viral RNA.
– If DNA-free RNA is required, digest either the sample or the eluate with RNase-free DNase. DNase in the eluate must be inactivated by heat treatment (15 min, 70°C)

Clogged Column

1. Precipitates were not removed.
– When using plasma samples, remove visible Cryoprecipitates by centrifugation for 5 min at 3000 × g

2. Lysate not completely passed through the membrane
– Centrifuge for 1 min at full speed or until all the lysate has passed through the membrane.

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RNSol H Reagent

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples which results in reduced RNA Yield.
– For better results, it is recommended to store sample in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Insufficient disruption and homogenization
– As a guide for a better disruption and homogenization, refer to sample preparation guidelines.
– Decrease the amount of starting material.

4. Incomplete pellet rehydration
– Pipette the sample repeatedly

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag.

3. Frozen samples used for stabilization
– For stabilization in RNaseLag, use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– RNSol H Reagent has been tested and is guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– Decrease the amount of starting material.
– As a guide for a better disruption and homogenization, refer to sample preparation guidelines.

2. Incompletely removed organic phase
– Do not pipette the entire aqueous phase, after phase separation.

3. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

4. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase Treatment.
– RNSol H pH might alter during storage. Check the RNSol H pH it should be around 4.2.

5. Protein contamination
– Remove the aqueous phase precisely.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. Contamination from inter or down phase.
– Be careful while pipetting aqueous phase and just draw off upper phase.

Not performing well in downstream application

1. Ethanol carryover
– Before rehydrating the RNA pellet, let Ethanol to evaporate and air-dry the pellet.

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RNSol Reagent

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples which results in reduced RNA Yield.
– For better results, it is recommended to store sample in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

3. Insufficient disruption and homogenization
– As a guide for a better disruption and homogenization, refer to sample preparation guidelines.
– Decrease the amount of starting material.

4. Incomplete pellet rehydration
– Pipette the sample repeatedly

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag.

3. Frozen samples used for stabilization
– For stabilization in RNaseLag, use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– RNSol H Reagent has been tested and is guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– Decrease the amount of starting material.
– As a guide for a better disruption and homogenization, refer to sample preparation guidelines.

2. Incompletely removed organic phase
– Do not pipette the entire aqueous phase, after phase separation.

3. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease-free water with pH ≥ 7.5.

4. DNA contamination
– Follow precisely the respective protocol, If RNA purification is still problematic further do DNase Treatment.
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

5. Protein contamination
– Remove the aqueous phase precisely.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. Contamination from inter or down phase.
– Be careful while pipetting aqueous phase and just draw off upper phase.

Not performing well in downstream application

1. Ethanol carryover
– Before rehydrating the RNA pellet, let Ethanol to evaporate and air-dry the pellet.

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Small RNA technologies

MiRjia Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.
– For better results, it is recommended to store samples in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag, Refer to page 9.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, based on sample type refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease free water with pH ≥ 7.5.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

3. Check RNSol pH
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeds kit specifications
– Refer to specifications to determine if the amount of starting material falls within kit specifications.

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Mirjia Lipid Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.
– For better results, it is recommended to store samples in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag, Refer to page 9.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, based on sample type refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease free water with pH ≥ 7.5.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

3. Check RNSol pH
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeds kit specifications
– Refer to specifications to determine if the amount of starting material falls within kit specifications.

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MiRJia PB Kit

Low yield

1. Inappropriate sample storage condition
– Avoid freezing and thawing of samples, which results in reduced RNA Yield.
– For better results, it is recommended to store samples in RNaseLag.

2. Incomplete cell lysis
– Too much starting material results in low RNA yield. To optimize the results, refer to Table 2.

Degradation

1. Too thick sample for stabilization
– Cut large samples into slices less than 5 mm thick for stabilization in RNaseLag.

2. Improper sample storage
– It is suggested to store samples in RNaseLag, Refer to page 9.

3. Frozen sample used for stabilization
– For stabilization in RNaseLag, Use fresh samples.

4. Storage duration in RNaseLag exceeded
– Refer to Table 4.

5. RNase contamination
– All buffers have been tested and are guaranteed RNase-free. RNases can be introduced during use. Refer to Appendix 1 for more information.

Low 260/280 ratio

1. Insufficient disruption and homogenization
– As a guide for better disruption and homogenization, refer to sample preparation guidelines.

2. RNA was diluted in low pH water
– Use 10 mM Tris-HCl with pH ≥ 7.5, or nuclease free water with pH ≥ 7.5.

DNA contamination in downstream application

1. No DNase treatment
– Perform DNase treatment.

2. No incubation with TWB1
– Incubate the spin column for 5 min at Room Temperature after addition of TWB1 and before centrifuging

3. Check RNSol pH
– RNSol pH might alter during storage. Check the RNSol pH it should be around 4.2.

Not performing well in downstream application

1. Ethanol carryover
– Preform another centrifugation before rehydration step to ensure no remaining of Ethanol on column.

2. Salt carryover
– Between washing steps, eliminate remaining flow-through from the rim of collection tube, by blotting on clean paper towels.

Clogged Column

1. Maximum amount of tissue exceeds kit specifications
– Refer to specifications to determine if the amount of starting material falls within kit specifications.

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Molecular Diagnostics

HLA Disease

HLA B-5 and B-51, B-51, B-5 Diagnostic Kit

DNA “smear “in the gel tracks; Background bands; Nonspecific PCR products

1. DNA concentration problem
– Check the DNA concentration is neither too high nor low. Aim for between 50 – 100ng of DNA per reaction.

2. Impure or degraded DNA
– Measure OD260/OD280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary.

3. Contamination
– Check Negative Control. Check sample DNA and re-extract if necessary. Use filter tips.

4. Incorrect amplification conditions
– Check the thermocycler.

5. Primer dimer bands evaluated as positive specific reactions
– Check band sizes.

Weak bands; Suppression of reactions

1. Use of insufficient amount of DNA
– Double the amount of DNA (reduce dH2O in preparation); use around 100 ng DNA per PCR preparation

2. Impure or degraded DNA
– Measure OD260/OD280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary.

3. Master mix or Sample DNA not added to PCR Mix
– Repeat PCR.

4. Insufficient mixing of the DNA or reactions
– Dissolve DNA at 37-65°C. Vortex well after preparing the master mix.

5. Tubes not correctly sealed
– Check the seal of the tube.

6. Incorrect amplification conditions
– Check the thermocycler.

7. PCR inhibitors such as ethanol, hemoglobin, heparin, beads contained in preparation.
– Use EDTA or citrate blood as base material; (after the DNA pellet has been washed with ethanol, ensure that it is sufficiently dry).
8. pH value of the DNA solution is too acidic (PCR cocktail changes color after adding DNA)
– Precipitate DNA once more and dissolve in nuclease free water.

9. PCR product leaked out of gel pocket
– Correct gel preparation with straight pipette tips.

Bands in the PCR mix of the negative control

1. By mistake DNA was pipeted into the mix of the negative control
– Possibly repeat preparation or make a note of it in the evaluation documents

2. Contamination of the reagents
– Exchange reagents

Amplification pattern is not interpretable

1. Incorrect interpretation of an artefact as a specific band
– Check the specific Interpretation Tables for correct band size.
– Check if all specific amplifications are correct in size or if an artefact (carry-over, primer dimer) has been misinterpreted as an amplification

2. Reactions loaded in the incorrect order
– Check alignment of PCR and gel lanes.

3. Individual PCR failure
– Check all internal positive controls are present. Reinterpret without any missing reactions.

4. Small amplicons missing
– Electrophoresed too far, small amplicons have run off the end of the gel, or past the ethidium bromide front, or are dispersed by entering preceding gel well. Use electrophoresis conditions suitable for your gel system.

5. New allele identified in sample
– New alleles may occasionally be discovered that may give rise to an amplification pattern that does not correspond to an existing allele(s). Please contact ROJE Technical Support Team.

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HLA B-27 Diagnostic Kit

DNA “smear “in the gel tracks; Background bands; Nonspecific PCR products

1. DNA concentration problem
– Check the DNA concentration is neither too high nor low. Aim for between 50 – 100ng of DNA per reaction.

2. Impure or degraded DNA
– Measure OD260/OD280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary.

3. Contamination
– Check negative control. Check sample DNA and re-extract if necessary. Use filter tips.

4. Incorrect amplification conditions
– Check the thermocycler.

5. Primer dimer bands evaluated as positive specific reactions
– Check band sizes.

Weak bands; Suppression of reactions

1. Use of insufficient amount of DNA.
– Double the amount of DNA (reduce dH2O in preparation); use around 100 ng DNA per PCR preparation

2. Impure or degraded DNA
– Measure OD260/OD280 (a quotient of 1.8 is optimal) and evaluate on agarose gel. Reextract DNA if necessary.

3. Master mix or Sample DNA not added to PCR Mix
– Repeat PCR.

4. Insufficient mixing of the DNA or reactions
– Dissolve DNA at 37-65°C. Vortex well after preparing the master mix.

5. Tubes not correctly sealed
– Check the seal of the cover strips or PCR mat (use press-on mat).

6. Incorrect amplification conditions
– Check the thermocycler.

9.PCR product leaked out of gel pocket
– Correct gel preparation with straight pipette tips.

Bands in the PCR mix of the negative control

1. By mistake DNA was pipeted into the mix of the negative control
– Possibly repeat preparation or make a note of it in the evaluation documents

2. Contamination of the reagents
– Exchange reagents

Amplification pattern is not interpretable

2. Reactions loaded in the incorrect order
– Check alignment of PCR and gel lanes.

3. Individual PCR failure
– Check all internal positive controls are present. Reinterpret without any missing reactions.

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PCR & RT-PCR

RT-PCR

nRT-ROSET , RT-ROSET Kit, Recombinant M-MLV

No PCR Product

1. Handling error
– Repeat the PCR carefully.

2. Too high incubation temperature
– Reverse transcriptase should be done at 42°C. Check the temperature of your heating block or water bath or your thermal cycler.
3. Starting sample degraded
– If RNA degraded, the RNA integrity may be affected.

4. Problem with starting sample
– Check starting material quality and storage concentration and quantity. Repeat the PCR with new dilution.

5. Not optimal primer concentration
– Repeat the PCR with different primer concentrations.

6. Not optimal Mg2+ concentration
– Repeat PCR condition with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM increments) using a 25 mM MgCl2 solution.

7. PCR Program
– Optimize PCR condition by changing annealing temperature.

8. Too short extension time
– Increase the extension time by increments of 1 min.

9. Not professional primer design
– As a guide refer to Appendix 3.

10. Problems with thermal cycler
– Check the power that the thermal cycler has been correctly programmed.

Smear PCR product

1. Too much starting material
– Make serial dilutions of sample. and perform PCR using serial dilutions

2. Too much enzyme concentration
– Use 2.5-5.0 units of Taq DNA Polymerase per 50-µl reaction

3. Not optimal Mg2+ concentration
– Perform PCR with different final concentrations of Mg2+ from 1.5– 5.0 mM.

4. Not professional primer design
– As a guide refer to Appendix 3.

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Product FAQ & Order FAQ

You can find answer of your question here

Order FAQ

Due to the commitment and efforts of our team to achieve customer satisfaction, easy ordering and secure shopping are provided to customers.

Order FAQ

1. what payment methods are accepted?
Your payment can be done in 2 forms:
– Online Payment; which is paid through the bank portal
– Or by depositing your money to the Saderat bank account number 0107346524006.(A bank account called ROJE Biotech Manufacturing Group) and then send the scanned payment slip to [email protected]

2. Is it possible to cancel or change the product after ordering?
– If your replacement or cancellation of the order does not interfere with the company’s production process, then replacement or cancellation of the order is possible. It is emphasized that if you need advice on product selection, contact our technical support at (+98)35-37237122

3. What are the payment conditions ?
– For individual the payment is in cash and before delivery and for legal persons according to the pre-concluded contract.

4- How soon can I expect my order?
– If the ordered item is in stock, it will be delivered within 24 to 48 hours and if not available contact the company to find out when the product will be ready.

5. How do I find out if my order is successfully registered via email or website?
– Once you have registered your order, we will send you an email with your order registration. Then, wait for our call for up to 24 hours. If you do not receive a call within 24 hours, call (+98)35-37237122

6. Can I order a product that is not in the product list?
– Yes, it depends on the product you are looking for. Call (+98)35-37237122 to stay informed of the process.

7. Is it possible to place an order by email?
– Yes, you can submit your order by filling out the ordering form and mail it to [email protected]

8. Is it possible to place an order by phone?
– Yes, you can submit your order by calling (+98)35-37237122 from 9:00 to 17:00. To do this, you must be sure of the catalog number of products in question. Refer to the products section of the site for necessary information.

9. How is the product ordering through the site?
– You can select the product category, product type, and volume you want by visiting the Product section, and then click Add to cart to add the product to your cart. If you order more than one kit, click on the Add to cart option. To see product prices, see the Cart section at the top of the page to see the price of your chosen product.

10. I need help for choosing a product. Who should I contact?
You can ask your questions to the ROJE specialists by calling (+98)35-37237122 from 9:00 am to 5:00 pm. And for outside office hours you can:
• Fill out the support form on the site or
• Email [email protected] and ask your questions.

Didn't find your answer?

If you didn’t find your questions through the list above, you can ask your questions in the list below.
Our specialists will answer you as soon as possible.

This post is also available in: Persian

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