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In this segment we have tried to answer the more usual, and challenging questions raised by our clients. Moreover, we put some possible reasons with appropriate solutions right below.
The absorbance ratio 260/280 is an indicator of the purity of isolated DNA. A value of 1.8-2 is generally considered pure for DNA.
If the ratio is lower than the expected range, it may indicate incomplete removal of protein contamination in the sample that have absorbance close to 280 nm.
The other possible reasons for this issue among users are residual ethanol because of skipping the drying step, low concentration and purity of ethanol used in the procedure, and reduced proteinase K activity because of improper storage of enzyme after preparation. Considering the volumetric ratios of starting sample to lysis buffer and ethanol is strongly recommended.
If the ratio of 260/280 is higher than the expected range (1.8-2), it suggests strong absorbance at 260 nm due to the addition of DNA carrier to the isolation procedure and improper cleaning of the pedestal arm of the nanodrop.
The absorbance ratio 260/280 is an indicator of the overall purity of isolated DNA. A value of 1.8-2.5 is generally considered pure for DNA.
If the value is lower than the expected range, it may indicate guanidine salts have been carried over during the elution step that has strong absorbance at 230 nm. To avoid salt contamination, apply wash buffers respectively as mentioned in the protocol, repeat the washing step with WB2 according to the procedure, while transferring liquids, avoid touching inside the column with the pipet tip, and avoid transferring bubbles formed in the lysis step.
Considering the volumetric ratios of starting sample to lysis buffer and ethanol is strongly recommended.
If the ratio of 260/230 is higher than the expected range (1.8-2.5), it suggests strong absorbance at 260 nm due to the addition of DNA carrier to the isolation procedure and improper cleaning of the pedestal arm of the nanodrop.
- Low quality of starting samples due to repeated freeze/thaw cycle significantly influences the quality of samples. Repeat sampling and use fresh ones.
- Incomplete lysis; incubation time and temperature duration in the lysis step are critical.
- Reduced proteinase K activity due to improper storage of enzyme after preparation.
- Using recommended alcohol in the protocol increases the efficiency of the isolation procedure.
BehPrep Viral RNA is suitable to isolate viral RNA, but it is not customized to extract viral RNA from urine.
It is not recommended to use BehPrep Genomic DNA to extract DNA from amniotic fluid.
BehPrep G-Plus DNA is suitable for DNA extraction from CVS. Before starting the procedure, wash CVS sample in PBS solution several times to separate maternal blood.
- The buffer you are using as a blanking solution must be the same as the buffer you used to suspend the DNA you are measuring.
- Try to remove ethanol as much as possible. Do not skip the drying step as described in the protocol. to complete removal of ethanol, open the column lid for 5 minutes to air dry the membrane.