The BioTechniques Molecular Biology Forum brings scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the DNA and General PCR Methods forum.
Q: My PCR products showed thin bands on a 1% agarose gel. Following gel purification with a Qiagen kit, I quantified the DNA using a Nanodrop spectrophotometer, which showed concentrations of only 5-9 ng/uL. I want to send these samples for sequencing, but I need at least 50 ng/uL of purified PCR product. How can I improve the amplification so that I can purify higher concentrations of products?
A: A 50 ul PCR normally produces around 1-4 ug of DNA. You should alter the PCR conditions to increase your yield. What are you using as a template? Poor yields from a primary PCR from RNA may indicate low expression, which can be difficult to improve. Nested PCR…
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