Direct Cloning of Blunt-end PCR Fragments. HELP !
Simon E Plyte
splyte at ocicl.oci.utoronto.ca
Fri Jul 1 10:29:23 EST 1994
In article <tsichlis-010794080620 at thalis.rm.fccc.edu>,
tsichlis at thalis.rm.fccc.edu (Tsichlis Lab) wrote:
> In article <2v1026$s69 at mserv1.dl.ac.uk>, mjones at crc.ac.uk (Dr. M.D. Jones)
> > Hi netters
> > I suspect that this may be a FAQ. But never mind.
> > I am trying to clone blunt-ended PCR fragments into SmaI cut M13 vectors.
> > So far no luck. Using the same batch of vector, T4DNA ligase and competent
> > cells, I can successfully clune AluI digested lambda DNA fragments.
> > The PCR fragments are 300 - 2000 bp in size. I clean them up by PEG pption.
> > This is a great method and we use it all the time to clean-up PCR fragments
> > for ABI DYE-terminator cycle sequencing. The original reference to this
> > method is Rosenthal et al. (1993) Nucleic Acids Res. vol. 21. 173-174.
> > The PCR fragments can be digested with restriction enzymes. I blunt-end the
> > fragments with T4DNA polymerase (+/- Klenow).
> > I have not used T-tailed vectors, as I would like to get blunt-end PCR
> > fragments to clone into SmaI cut vectors.
> > Any help, advice, suggestions, moral support, immoral support, URGENTLY
> > neede to keep my insanity intact.
> > Mick
I do a method similar to Lee which works pretty well:
Firstly I remove the oil with Diethylether. Then take 40 ul of the PCR
reaction and add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM)
and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37
for 30 min.
Then Phenol/chloroform extract
chloroform/IAA extract and then EtOH precipitate
you can then just "shotgun" clone this DNA. However, if you have several
non-specific bands then you may want to gel purify the fragment first.
One point to note....
We have given up blunt end cloning into Sma I sites where possible.
Several people have reported problems with Sma I cut DNA. By choice we
clone into EcoRV sites.
Hope this works for you (or Lee's method).
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