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Direct Cloning of Blunt-end PCR Fragments. HELP !

Dr. M.D. Jones mjones at crc.ac.uk
Fri Jul 1 06:52:38 EST 1994



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

 
Mick Jones 
Department of Virology
RPMS,  Du Cane Road
London,  W12 0NN
Tel: 081-740-3328
Fax: 081-743-8331
email: mjones at rpms.ac.uk

"Smoke me a kipper, I'll be back for breakfast"
Ace Rimmer (Red Dwarf)



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