IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

Direct Cloning of Blunt-end PCR Fragments. HELP !

Tony Hodge tph at mrc-lmb.cam.ac.uk
Mon Jul 4 08:22:08 EST 1994


Subject: Direct Cloning of Blunt-end PCR Fragments.  HELP !
From: Dr. M.D. Jones, mjones at crc.ac.uk
Date: 1 Jul 1994 12:52:38 +0100
In article <2v1026$s69 at mserv1.dl.ac.uk> Dr. M.D. Jones,
mjones at crc.ac.uk writes:
>
>
>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)


Dear Mick,

I routinely clone my PCR fragments blunt ended into pUC in the first
instance before checking the sequence and subcloning into eg
expression vectors.  I do a standard PCR with a 5minute at 72C step
at the end.  I then klenow the PCR product, clean it up either by
geneclean or  a gel followed by geneclean (or Mermaid if its too
small) and then ligate into HincII cut pUC.  Note the HincII which I
find is more reliable since SmaI can nibble at the blunt ends of
your vector.

Note I do note try to extract the oil before Klenow.

Alternatively you could use NEB's Vent Polymerase which gives blunt
ends directly and less errors, but watch out for the 3'-5'
exonuclease which can cause some problems especially if you have too
much missmatch near the ends of your primers.

In both cases watch out for high residual concentrations of dNTPs as
this can inhibit blunt ended ligation.  I always clean with
GeneClean or run on a gel and Mermaid the bands before cloning for
this reason.



Tony



.
Tony P Hodge
Structural Studies Division
Medical Research Council Laboratory of Molecular Biology
Hills Road
Cambridge
CB2  2QH
UK

Phone (0223)  402260

Fax     (0223)  213556



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net