Biotinylated oligo ligation to lambda DNA
wag22 at cam.ac.uk
Wed Nov 22 19:50:21 EST 2000
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). 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. I then clean it with a Microspin S-400 column to get rid of the unound oligo and then cut the product with BgLII enzyme to yield a 400 bp biotinylated double-strand DNA. When I run this on a gel and blot it with anti-biotin and BCIP I cannot detect anything. (I can detect the pure oligo B though). I tried to detect it by detecting binding to a Streptavidin Chip in a BiaCore, but this also didn't work. Can you please give me a protocol for binding this biotynilated oligo to the lambda DNA. I use standard buffers and enzymes at their prescribed temperatures, but after many tries I cannot get it to work.
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