HELP WITH LIGATION OF PCR RESTRICTION FRAGMENTS
VMiao at oregon.uoregon.edu
Thu Aug 26 10:11:21 EST 1993
> kanemats at angis.su.oz.au wrote:
> : WANTED: PROTOCOLS FOR LIGATING RESTRICTED PCR FRAGMENTS
> : I would like any successful protocols available which
> : could allow the ligation of 3 PCR products (each of
> : approximately 200bp) after restriction digestion by PstI
> : (PCR products 1 & 2) or by BamHI (PCR product 2 & 3).
Can you do the ligation in 2 steps, checking for the correct
product from 1+2 by PCR, then gel isolating (or whatever) that product and
using that in a ligation with 3? This is really a sort of control for
shooting your 3-piece ligation, because you don't yet know (at least you
say in your post!) whether you know that all three components are
for ligation. It wouldn't be a lot of extra work, and if the first step
you are already half way towards making your product anyway, so your
wouldn't be wasted.
I tried a similar 3 piece ligation/selection and it ended up working in
steps anyway, as I tried to figure out why, oh why, my darn-near perfect
scheme (!) didn't work! So, ....
Ligate 1 and 2. If you get a 400 bp PCR product (1+2),
then you know the ends, e.g. Bam, are OK. Save this product.
Ligate 2 and 3. If you get a 400 bp PCR product (2+3),
then you know the ends, e.g. Pst, are OK. Save this product.
Then ligate (1+2) with 3 and PCR, and in parallel (might as well ....!)
ligate (2+3) with 1 and PCR. ..... something should work right!
In the meantime, do check enzyme ends and the a newsgroupFAQ
as suggested by Brian Foley!
(Incidentally, I used 20 ng each fragment, 0.1 Weiss Units of ligase in a
10 microliter ligation reaction incubated at room temp for 1 hour.
I then used 2 microliters for PCR).
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