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MEGAPRIMER PCR

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MEGAPRIMER PCR

Postby Andres » Wed Jan 25, 2012 5:21 pm

Hi!

I´m trying to join two DNA genes in order to create a chimeric one. I´m using the megaprimer methods. The templates are one of 930 pb and the other 700pb. The have 15nt in common. I have try different Tannealing from 58-68 and i found a condition where i obtein a band in near the expected MW but i have really poor yield...I purified the product and try doing a 3rd PCR with the flanking primers, but i had no success. Do you have any advice? may be 15 nt is not enough and i have to add more nt in common, what do you think?

Thanks for your commets

Andres
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Postby JackBean » Thu Jan 26, 2012 9:39 am

20-25 nt should work better. Shouldn't you add the flanking primers into first PCR as well?
http://www.biolib.cz/en/main/

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Postby Andres » Thu Jan 26, 2012 1:54 pm

Dear Jack
I was adding only the reverse primer in the first PCR...Yesterday I try adding both flanking primers and now I´m running this....I´ll tell you how it works promptly...if I have no success i´ll will add more nt for the region in common in order to make the annealing region more stable....Thanks for reply!

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Postby JackBean » Thu Jan 26, 2012 2:25 pm

let's say it looks like this
________..............
........____________
(dots are nothing, line is your DNA)

so you were adding primer like this:
________............_
........____________
(the red line)
so only on one side? In such case, you will have only linear amplification, thus lower production.
http://www.biolib.cz/en/main/

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Re: MEGAPRIMER PCR

Postby Andres » Tue Jan 31, 2012 5:38 pm

Dear jack

i tried adding both flanking primers but i obtained a broad band
Image. I purified it and did a 3rd PCR with both primers, but did not have any amplification....

Now I order 2 more primers in order to extend the overlaping region to 75nt

One more thing, In the Megaprimer method, I did the first 5 cicles with the template and the Megaprimer, and then, in the annealing step, I add both flanking primers...i have read different protocols that use the same aproach.
I really don´t know what´s happening....

thanks for your Help!

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Postby canalon » Tue Jan 31, 2012 7:36 pm

What pollymerase are you using? I once tried megaprimer PCR and could not get it to work with regular Taq, and when I switched to Phusion (from EB) It worked much better. Not the cheapest polymerase, and I am sure that other high fidelity mix can do as well, but maybe you can try one of those.

And by the way, it did not work for all of my experiment, so I know that this not a failsafe advice, just something to consider...
Patrick

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Postby Andres » Tue Jan 31, 2012 8:11 pm

In my last experiments i used a regular Taq pol from sigma because i´m trying to find out a condition ( T anealing, [Megaprimer] and [Template] to obtain the fusion. However, I have tried at the begining with a pfu also from sigma and I didn´t have a good result. I´ll consider using another Hi-Fidelity polimerase and i tell you how it works...

Thanks!

Andres.
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Postby canalon » Thu Feb 02, 2012 2:56 am

I See and understand. The worst is that you have to retry the conditions when changing the polymerase as they can behave quite differently. Or maybe the larger overlap will solve your problems anyway.

Good luck
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