re-amplification of a PCR product - is it recommended? (May/05/2009 )
hi all,
I need a lot of PCR product for my cloning experiments and unfortunately my PCR product is not enough. Actually I did an RT-PCR from an RNA sample but that sample is now all finished.
the only way for me is to use my own PCR product to get a final 300 ul PCR product.
my friends say re-amplification of a PCR product might give mutations. the size of my product is 1.7 kb.
I use Tfl plymerase from Promega. why is it risky to do that?
Curtis on May 5 2009, 01:45 AM said:
I need a lot of PCR product for my cloning experiments and unfortunately my PCR product is not enough. Actually I did an RT-PCR from an RNA sample but that sample is now all finished.
the only way for me is to use my own PCR product to get a final 300 ul PCR product.
my friends say re-amplification of a PCR product might give mutations. the size of my product is 1.7 kb.
I use Tfl plymerase from Promega. why is it risky to do that?
I don't know why it is risky but receltly i have cloned and sequenced a promoter sequence about 2kb in size. Actually first i used genomic DNA to amplify but the PCR product was not enough after gell purification.And somehow i lost the genomic DNA and i was too lazy to extract it again. What i did was that i used the PCR product as template and re-amplified the gene and then after T-Vector cloning, sent out for sequencing. The sequence i got was exactly same as reported before. So it worked for me and i hope it would be good for you too
. And if you got any information regarding the risk attached with re-amplification then do tell me.
thanks
I don't think re-amplification is a problem...It depends upon the fidelity of the enzyme. Use a good enzyme and everything is okay.
Best,
TC
yeah, enzyme is also important...I think Fermentas has a Pfu polymerase which is proofreading...mine is Tfl....however I'm going to order a high fidelity master mix from Fermentas next week.
Curtis on May 5 2009, 12:45 PM said:
I need a lot of PCR product for my cloning experiments and unfortunately my PCR product is not enough. Actually I did an RT-PCR from an RNA sample but that sample is now all finished.
the only way for me is to use my own PCR product to get a final 300 ul PCR product.
my friends say re-amplification of a PCR product might give mutations. the size of my product is 1.7 kb.
I use Tfl plymerase from Promega. why is it risky to do that?
You may try to use high-fidelity enzymes with high processivity.
Like PFU ultra or Pfu turbo - both from Stratagene
If you use a proofreading polymerase, be aware that TA cloning won't work, as the polymerases don't add an A overhang like Taq does. I'm using Phusion from Finnzymes at the moment, it seems pretty good.
bob1 on May 5 2009, 04:45 PM said:
Thank you bob
I should add that to get an A overhang:
1) Purify your PCR product to remove the proofreading polymerase, so that it won't snip off the A after it is added.
2) Add some dATP and PCR buffer (including Mg2+ to your purified product.
3) add some Taq.
4) incubate at 72 degrees for 15 minutes.
A overhangs added.