Biotinylated oligo ligation to lambda DNA
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Nov 23 04:48:23 EST 2000
In article <000a01c054df$7c897fc0$c3d16f83 at cam.ac.uk>, the eminent A
Germishuizen at BIOSCI/MRC Human Genome Mapping Project Resource Centre
> A few months ago I ordered a 3` biotinylated oligo (12 bases long,
> plus 4 T spacers) and I have tried to ligate it to a linearised
> lambda DNA (40 kbp).
What do you mean by linear lambda. Do you mean full length 48502bp or
cut lambda such that you have a 40kb fragment?
>First I add a phosphate to the 5` end of the
> oligo with T4 Kinase. Then I linearise lambdadna by heating it. I
> then anneal the two together for 16 h, followed by ligation with T4
> ligase for 16 h
Assuming you have full length lambda then I presume you have an oligo
complementary to the cos ends? When you heat the lambda do you have the
oligo in there in excess? I take it you heat it up in a buffer with some
NaCl, if it is water lambda DNA will just fall apart and give umpteen
fragments. Do you then leave the heated beaker of water to cool down to
room temp and then set up the ligation?
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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