Minimum number of PCR cycles to see a product? - (Mar/09/2009 )

Dear all,

I am making a mutant library using biased dNTP and MnCl2. The higher the number of cycles, the higher the number of mutations. I have done an error-prone PCR of 10 and 15 cycles before and they gave me too many mutations. So, I am trying out 5, 7 and 9 cycles. I have positive controls, in which normal dNTP is used and no MnCl2 is used at all. It undergoes the same PCR conditions of 5, 7 and 9 cycles. When I run my positive contrls on the gel, I see the desired PCR products at the right sizes. But my supervisor told me that normally, we wouldn't see a PCR product with 5 cycles because that number of cycle is too low. Has anyone seen PCR products on the gel after 5 cycles? Please advise me.

Thank you in advance!

This will depend crucially on the amount of template DNA. Under ideal conditions, five cycles will amplify 32 times, so if you start with sufficient DNA, then indeed you will see amplification after five cycles. This is probably where you want to be working for your purposes.

phage434 on Mar 10 2009, 11:09 AM said:

Dear phage434,

Thanks a lot for your reply. No wonder my supervisor was telling me that maybe I added too much template. I have already checked and I added 25ng of template, which was what was stated initially. And, 32 times was derived by 2^5. So if I have like 100 DNA molecules, it means it will be amplified 32 times for each DNA molecule. Right?

Perfect PCR is an exponential amplification. -- after every cycle, the amount of product is doubled (assuming 100% reaction efficiency). If all 100 double stranded molecules were amplified through 5 cycles, you'd have 3,200 double stranded copies.

If you need lots of PCR product for cloning, but only limited mutation rate I would suggest:

1) Use error-prone PCR (with Mn) for a limited number of cycles say 4- 8 with ca 25 ng template.
2) Add 1 µl of the PCR product to 99 ul of you standard PCR mix (Mg only) and do a second amplification for 18-25 cycles.

Hope this helps

HomeBrew on Mar 10 2009, 09:24 PM said:

Dear HomeBrew,

Thanks a lot for clarifying my doubts! That really helped!

klinmed on Mar 11 2009, 02:36 AM said:

Dear klinmed,

Thanks a lot for your reply. What you suggested was similar to what I did! I actually did an error-prone PCR with Mn and biased dNTP for 5, 7, 9, 10 and 15 cycles with 25ng of template. After that, I did a Dpn1 digest before I took 1ul of the PCR product to 30ul of PCR mix for a re-PCR for 20 cycles! Now, I just need to characterize the quality of my mutated libraries.