Blunt lig. help (NL)

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Thu Sep 28 17:27:06 EST 1995


In <43vb7u$fir at mercury.IntNet.net> dbois at netsrq.com writes:

> A.A.vanApeldoorn at med.ruu.nl (Aart A. van Apeldoorn) wrote:
> >Thanks for your suggestions, however i tried bluntligation with and
> >without PEG8000, but this seems to make no difference the results are the
> >same. I also use a fresh stock of ATP each ligation so this can't be the

SNIP

> A method for cloning PCR products by blunt-end ligations was published
> in BioTechniques last year.  It works very well for me. It really is
> quite simple and fast.  No buffer changes, either! 
> 
>  Here's the reference:  Kai Wang, Ben F. Koop and Leroy Hood  "A
> Simple Method Using T4 DNA Polymerase to Clone Polymerase Chain
> Reaction Products", BioTechniques, vol. 17, no.2, pp.236-237, 1994.

I wholeheartedly "second" Danuta's suggestion.  This technique is one of 
the very few that worked the first time for me.  However, I have found some 
perhaps obvious limitations.  First, from my experience, the PCR reaction 
must be a pristine reaction - that being, no spurious bands or smears.  
If it isn't a pristine reaction, you have a whole slurry of ligatable 
"stuff" most of which isn't what you want.  Only on optimized PCR reactions 
with defined products has this method worked for me.  Second, I have not 
been able to succeed in ligating a purified fragment in which I took a 
portion of the fragment and added all the reagents, including Taq buffer 
and enzyme, and go through the protocol as if the fragment had been PCR'ed. 
 This latter point is one that I would like to overcome as it would help 
many a ligation.

On the PCR (actual product) where this protocol worked, it worked like 
greased lightning.  I took a sample of the kinased product - per the 
protocol and basically dropped it into pBluescript cut with EcoRV which had 
been alk-phos treated.  It was one of the easier ligations that I had ever 
conducted recently.

I hope this helps,
David





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