how do I get rid of adapters / linkers after ligation to my cDNA

John Wertz azjew at imap1.asu.edu
Tue Nov 9 20:53:35 EST 1999


The only technique which comes to mind is gel filtration.  Princeton
Separations, Inc. sells a product called CENTRI-SPIN-40.  This is a spin
column for a microcentrifuge which allows larger DNA products > 150bp to
pass through while retaining nucleotides, buffer salts, and short (<25-mer)
oligonucleotides. The problem with this is your linker dimer is to big for
efficient retention.

The only other idea I have come up with is if you treat your linker with CIP
(calf intestinal alkaline phosphatase) before you ligate it to your cDNA,
this will eliminate linker dimer formation.  Your cDNA+linker constructs
will have a nick in them where the 5’ phosphate was removed from the linker.
If you run your first few PCR cycles at a high annealing temperature taq
polymerase should "finish of the ends" of your cDNA+linker template.
Subsequent PCR cycles should be run at an annealing temperature appropriate
to your linker primers.

Good Luck :)



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