Ligation of PCR products
num208jn at MBDS.NRC.CA
Fri Jul 19 08:10:27 EST 1991
In article <9107182217.AA23884 at genbank.bio.net>, ST403161 at BROWNVM.BROWN.EDU (Michael Newstein) writes:
>I have read that it is exceedingly difficult to blunt-end ligate products from
> PCR (without cleaving first in internal restriction sites). I am attempting
>to clone the product of a PCR reaction into a blunt end cutter site (eg SMA 1)
>in pBluescript. Does anyone have suggestions?
I'll post this to the newsgroup because it is a question which comes
up from time to time.
The following worked for me:
After the PCR reaction, I added 10 units of T4 DNA polymerase to the
sample (I didn't remove the oil, one has to careful to ensure the
sample + T4 pol is well mixed). I incubated it at 37 deg for 15 min,
and then did a regular clean up and agarose gel/glass milk excision of
the fragment. I find that if I heat inactivate the T4 pol, it gives
poorer yields... maybe going up to 70 deg sends the exo activity nuts
after the pol activity has quit... who knows.
There is also a methods paper (in Nucl Acids Res , I believe) that
recommends a proteinase K treatment of the PCR'd DNA. The authors say
that this treatment improves cloning ability of PCR products... I
haven't tried it.
By the way, when I cloned my 650 bp PCR product, the positive clones
were all light blue.... I was cursing because the transformation
plates had few white colonies.
Hope this helps.
John Nash, Internet: num208jn at mbds.nrc.ca
Institute for Biological Sciences,
National Research Council of Canada, Ottawa, Canada K1A 0R6.
Disclaimer: All opinions are mine, not NRC's!
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