I started running nested pcr this week using Extract N Amp. This is my first time doing this, and after the second reaction, my water negative showed positive bands. I will look into contamination issues, but the DNA based negatives were negative. My questions are about how you are supposed to run the second round pcr:
1. Do you usually dilute the first round products? (1:100 is what I have seen being used)
2. Do you clean up first round products?
3. Are you supposed to use the Water negative products from the first round in the second round, or fresh water?
Thanks!
Dear Kristen,
1. I usually dilute the first PCR products about 1:10 since Nested PCR is as very specific reamplification.
2. Here, we don't use any purification method for the first round products, just for more trustworthy results.
3. And yes, it's recommended to use the first negative control product for Nested. You can also use fresh water only to prove your reaction is pure, but always use the first negative control too.
I hope I've helped.
dilution 1:100 without purification is best for your nested PCR. It is almost impossible to prevent contimination in the water control, if you succeed in avoiding this you are a top investigator. So you should keep this in mind when analysing the results, but most times it will not influende your results because the contaminant is present only in a few copies while your positive sample will contain much more copies normally.
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