small insert ligation

John Watson watson_j at bms.com
Thu Sep 5 08:16:24 EST 1996


Morello Jean-Pierre wrote:
> 
> I'm trying to insert a 200 bp fragment into a 3.4 kb vector using two sticky
> site enzymes (Bam HI and HinDIII). The 200 bp insert comes from a PCR product
> which has sufficient nucleotides on each end to allow for enzyme digestion.
> 
> So far, I've tried to either purify both pieces of DNA prior to ligation (using the
> Geneclean kit from Bio 101) or I've taken DNA straight after digestion and
> phenol/chloroform extraction. In both cases I only get recircularized vector
> although I have verified that both enzymes have digested the DNA. In addition,
> I have treated the vector with alkaline phosphatase and phenol/chloroform
> extraction prior to digestion.
> 
> If anyone has experience with small insert (approx. 200-300 bp) ligation
> I would greatly appreciate the help.

I have done this often for fragments in the size range you describe, though not with 
those particular sticky ends.  Please check our Meth. Enzymol. chapter for detailed 
protocols:  

Wilkie et al. (1994). Meth. Enzymol. 237:327-344.

Good Luck.

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John Watson                                                          |
Bristol-Myers Squibb Co.                                             |
watson_j at bms.com                                                     |
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