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.
We use a method published in BioTechniques Vol.13, No.4 p.613.
I phenol extract the PCR product, ethanol precipitate it, then treat it for
1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4
DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl
pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.
I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front
of the band, pour in some 0.7% LMP agarose(BRL), run the product into it
The PCR product in the LMP can be used for ligations directly, without
purification. The ligations take place at room temp on the benchtop.
I prepare the vector with minimal digestion (~2hr) then treat it with
shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector
to have on hand, so I know it is good and will have a low background.
You may also want to try using an EcoRV cut vector instead of a Sma cut
Lee Grimes/Tsichlis Lab
Fox Chase Chase Center
7701 Burholme Ave
Phila, PA 19111
Grimes at thalis.rm.fccc.edu