pcr of large products

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haf2006
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pcr of large products

Post by haf2006 » Thu Apr 27, 2006 10:12 am

Hi

i have a vector of 10.5kb which i need to mutate. I have tried doing this in two stage pcr one peice up and ligating it into the vector without success. have now decided to try overlap pcr. however we still need to get two pcr products of 5.5Kb each. Does anyone have any advice on this? no one in our group has experience of doing pcr on such large DNA template. Any help would be much appreciated.

so far we have tried standard pcr annealing at 55 and touchdown from 65 - 55 with an extention time of 5.5 minutes at 60C (needed due to AT richness in vector)

ta

haf :?

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LilKim
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Post by LilKim » Fri Apr 28, 2006 3:16 am

are you trying to do site specific mutagenesis? or random?

If you're trying to make a single BP change than can be done really easily b using "mismatched primers".

As far as your PCR reaction. .. you're going to need to extend for longer (the estimate is 1 minute per kb ... so, your extension needs to be at LEAST 10mins/cycle).

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Post by haf2006 » Tue May 02, 2006 9:54 am

Hi

i am trying to do a double base change and have tried an over 10 min extension but it doesn't work. this is why we were thinking of doing overlap pcr so the product was in two halves and could be joined together.

do we need to add more dNTP's than normal and more enzyme as we are worried they might run out with such a long pcr time?

thanks

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LilKim
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Post by LilKim » Tue May 02, 2006 10:50 pm

don't add more DNTP's that will throw off concentrations of the master mix.. you're better off doing smaller volumes and doing reactions in several tubes.

So ... I'm not sure if i'm understanding "double-base pair change"... there are several ways that that can be accomplished. If you're using primers that have 2 mismatched bases.. the primers (having the 2bp mismatches have to be very LONG and you'll have to lower your annealing temperature significantly. This may cause non-specific priming (however the legnth of the primers will make your reaction more stringent) If you have numerous bands you can "cut them out" and gel purify... or PCR again (raising the annealing temp 2 degrees).

I don't know if this info will be helpful.. cuz i'm not sure if you've given me enough info to fully understand what you're trying to do.

(... and, this is just a guess ... I too am learning molecular techniques)

buena suerte!
- KIM

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