Mutagenesis - large insertion

PC peter.cherepanov at REMOVE.uz.kuleuven.ac.be
Tue Jun 11 12:00:20 EST 2002


Design three sets of PCR primers, so that you will have three partially
overlapping PCR fragments (17 bp overlaps are enough), purify them on gel
(make sure there not to contamine the fragments with original DNA), mix and
amplify with the outer primers. Clone the final fragment where it needs to
be.
Use Taq or Expand for that (if you are lucky it might work with Pfu).
(I assume that you can amplify your 100bp insertion fragment from somewhere,
if you have to make it synthetic - use 2 overlapping oligos, anneal and
extend).

Peter


<mail2news at anon.lcs.mit.edu> wrote in message
news:20020611120921.32579.qmail at ww02.hostica.com...
> Mutagenesis - large insertion
> Yumiko Matsuoka 11.06.2002 05:09
>
> I have been trying to insert a large sequence (~100bp) using Stratagene
Exsite mutagenesis kit and have not had any luck so far.  Do any of
subscribers have any experience making such large insertion?  There is no
convenient restriction sites or possibility to insert restriction sites.
Any suggestions are appreciated.  Thank you.
>
>
>
> http://www.biowww.net/index.php/forum/message/26





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