How to get rid of excess adaptors?

John Dixon jpcd0 at mole.bio.cam.ac.uk
Thu Mar 2 11:40:25 EST 1995


Hi all,

I am trying to make a lambda library of mouse genomic fragments. I have
digested with various enzymes and added adaptors so that I can clone them
all into the same site in the lambda vector I want to use. I can see on a
gel that the adaptors (dephosphorylated) work as after their addition the
DNA will no longer religate, whereas unadapted DNA religates nicely.

Anyway now I need to get rid of the excess adaptors before I go any
further. I don't want to gel purify if I can help it, so what do you
recommend?

Can I use sephadex G50 spin columns and if so what is the nucleotide cut
off point? My adaptors are two 18mers annealed over a 14bp complementary
stretch, leaving a 4bp overhang. So 18mers must be retained but how much
of the small frags will stay in the beads?

Or am I better of using a PCR product purification set-up like QIAquick to
wash the primers away and then elute the DNA? 

Any suggestions? Thanks in advance

-- 
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



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