HELP WITH LIGATION OF PCR RESTRICTION FRAGMENTS

[Vivian Miao]

> 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).
 [stuff deleted] 

Hi Francisca,

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
trouble
 shooting your 3-piece ligation, because you don't yet know (at least you
didn't
 say in your post!) whether you know that all three components are
competant
 for ligation.  It wouldn't be a lot of extra work, and if the first step
works, then
 you are already half way towards making your product anyway, so your
effort
 wouldn't be wasted.  

I tried a similar 3 piece ligation/selection and it  ended up working in
two
 steps anyway, as I tried to figure out why, oh why, my darn-near perfect  
 scheme (!) didn't work!  So, ....

In parallel:

 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).