annealing - (Jul/18/2001 )
You could try purifying the correct double standed oligo by PAGE, then at leats you know it is the right oligo you are trying to clone and your problems ar with the enzyme digests etc.
I have constructed some oligonukleotides that contains three repeated sequences.I have annealed the sequenses , once at 70 dg C slowly by cooling it down,and once at 60 degrees for 2 minuits. Whwn the different oligos were annealed they were about 100bp and 70 bp. they were supposed to have the restriction sites Hind3 and Pst1. I also cut the vector pEGFP with these enzymes and ligated (T4 ligase) the vector+oligo over night at 16 dg C.I get no colonies after transfomation to dh5 competent cells? I have treid this several times, also using the vector pSL101-also no result. What Is wrong?Please give me some advise! Beate