Help on PCR cloning

Thomas Hansner Thomas.Hansner at rz.ruhr-uni-bochum.de
Wed Dec 11 09:59:45 EST 1996


On Mon, 09 Dec 1996 18:25:01 -0800, ramjam at scripps.edu (Ramanujam
Raman) wrote:

>Dear Netters,
>
>I am trying to clone a piece of promoter region using Linker PCR.  It is a
>700 bp fragment from a larger piece in BSKS.  I made PCR primers that
>would just give me this 700 bp fragment.  Both primers have linkers that
>includes the four base in the sequence GGCC followed by a restriction site
>sequence and then the actual primer sequence.  The forward primer includes
>a HInd III site and the reverse primer includes a Bam HI site.  
>
>The PCR reaction gave the product of the correct size with very litte
>primer dimers.  I have phenol-Chloroform extracted the PCR product and
>ethanol precipitated in the presence of glycogen.  I then used a fraction
>of this product for restriction digest with Hind III and Bam HI followed
>by ligation into the correspondingly digested BS vector.  Ligation was
>carried out at 16 C overnight and then transformation using Xl-1 blue
>cells from stragene.  
>
>So far I get no colonies or only very few colonies that happens to be WT. 
>I use insert to vector ratio of 4 or 5:1.  Has anybody out there tried PCR
>cloning successfully and if so, does any of the above step seems odd or
>inhibitory in any way to the cloning process?  Is it okay to clean the PCR
>product by Phenol-CHCl3?  Is it okay to use glycogen to precipiate the
>product?  Given the fact that there are 4 bases beyond the restriction
>site, does either Hind III or Bam HI inefficient in digesting at ends?  I
>would appreciate any help with respect to the above.  Please email your
>replies to my email address.  Thanking you in advance,
>
>RAM.

Hi !

How long do you digest your fragments with HindIII/BamHI ?
HindIII is known to be a little inefficient in digesting DNA fragments
at the end. For further details take a look at the NEB catalog (S.
238-239).

Tom



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