I’m carrying on RT-PCR and met some problems. Could anyone help me?
1. What problem will be caused by DNA contamination of RNA in RT-PCR?
2. What’s the optimal Mg2+ concentration in the PCR system using pfu
polymerase?
The problem of DNA contamination depends on your purpose. Quantitative measures like real-time PCR can’t be done with DNA contamination when you do RT – it skews the results. If you want to amplify some known coding sequences, it shouldn’t be a big deal. You can order most full-length clones, however.
There is no real optimal Mg2+ for PCR. Pfu will work over a wide-range of Mg2+ concentrations. It will not work with excessive amounts of Mg2+ or anything else crazy.
