NUM208JN@NRCCAD.NRC.CA (John Nash) (03/13/91)
RP>Can anyone help me with m13 cloning I have some problems
RP>with ligation a PCR product into m13 vector (m13mp18)
RP>The size of fragments is varying from about 100bp to
RP>300bp size in lenght. I have opened the m13 vector
RP>by pstI /HindIII double digestion and also I have
RP?di>tested these PCR products same way because we have
RP>planned primers so that PCR is (probably) producing
RP>pstI and HindIII sites to the ends or near of the ends
RP>of these fragments.... If anyone has tried to introduce
RP>DNA fragments into m13 vector please let me know of
RP>the problems and how did you solve those problems...
RP>Especially talking about PCR fragment cloning into
RP>m13 and also into expression vectors too...
I haven't tried M13 but have tried the following protocol using BRL's
phagemid (pUC derivatives). I cut the vector with HincII, and do a
standard pcr reaction to get insert DNA. After the pcr reaction,
separate the reaction from the mineral oil, add approx. 10 units of T4
DNA Pol, incubate it at 37deg for 30 min, and gel purify the fragment.
I ligate this DNA (1 or 2 ul) with the vector (25 ng), electroporate
into DH5alpha cells and plate for colonies on ampicillin/xgal media.
I usually get 100 to 200 colonies per plate with about 5% to 15% white
colonies. I ignore this, because in the case of two 600 bp pcr
products, the correct clone (identified by subsequent sequencing) was
light blue on xgal plates! I do colony lifts using the gel-purified
pcr product as a probe, and usually about 5 to 15 colonies (per plate)
give signals.
It has worked for me at least a half-dozen times. Another thing of
interest... yesterday, I ran across an NAR paper that was titled
"Improved cloning efficiency of polymerase chain reaction (PCR)
products after proteinase K digestion." by Crowe et al., 1991. Nucl.
Acids. Res. vol:19 No:1 page 184. The authors claim that Taq pol may
stick to the ends of pcr'd fragments thus reducing "clonability".
They claim that proK treating the fragments increases cloning
efficiency umpteen fold... sounds like I may need to try this next
time I have to clone a pcr product!
Hope this helps... if you need more details, email me directly.
cheers,
John,
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John H.E. Nash <Bitnet: NUM208JN@NRCCAD.NRC.CA >
Institute for Biological Sciences,
National Research Council of Canada,
Ottawa, Canada K1A 0R6.
Phone: (613) 990-0990 Fax: (613) 952-9092.
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