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re-amplification of a PCR product


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#1 Curtis

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Posted 05 May 2009 - 01:45 AM

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?

#2 Signal

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Posted 05 May 2009 - 02:23 AM

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?


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/we have to wait for 30 years to let the mirAcle haPPen....someone here told me!!!!

#3 T C

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Posted 05 May 2009 - 02:48 AM

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

#4 Curtis

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Posted 05 May 2009 - 04:02 AM

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.

#5 Michaelro

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Posted 05 May 2009 - 09:50 AM

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?


You may try to use high-fidelity enzymes with high processivity.
Like PFU ultra or Pfu turbo - both from Stratagene

#6 bob1

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Posted 05 May 2009 - 04:45 PM

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.

#7 Curtis

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Posted 05 May 2009 - 05:10 PM

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.


Thank you bob

#8 bob1

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Posted 06 May 2009 - 04:26 PM

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.




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