Some problems in RT-PCR

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schmyly
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Some problems in RT-PCR

Post by schmyly » Tue May 30, 2006 11:57 pm

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?

tranjo
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Post by tranjo » Sat Jun 03, 2006 4:01 pm

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.

Best, J

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