Shifting Reality

Dear All!

Unfortunately, I have problems with the ligation of LoxP sites...

The procedure I follow:

The annealing of LoxP oligos (the ds oligo has got overhangs) in PCR heat block:

- 5 ul FW ss olgo
- 5 ul REV ss oligo
- 10 ul 50 mM NaCl solution

The conditions:

95 C - 10 min.
90 C - 10 min.
85 C - 10 min.
80 C - 10 min.
75 C - 10 min.
70 C - 10 min.
65 C - 10 min.
60 C - 10 min.
55 C - 10 min.
50 C - 10 min.
45 C - 10 min.
37 C - 10 min.

I use 5 ng of vector (6200 bp) and the approprite amount of insert (40 bp) [I determined the amount with ligation calculator using 1:5 molar ratio.] Then I ligated 1,5 h in 15 ul final volume on 18 C.

I had success with annealing of other ss oligos (65-70 bp) and with ligation of these and larger fragments using these parameters and conditions. I don't know whether the problem is the wrong annealing or the wrong ligation...

Has anyone got any idea about the problem? A little help would be appreciated

Thanks,

SF