Dear all,
I want to ligate a ssDNA with a ~2-kbp dsDNA with a 15-nt overhang. The
ssDNA oligo has a 60-bp hairpin construct with a 15-nt overhang at 5'
end. These two 15-nt overhang on ssDNA and long dsDNA are designed to
complimentary to each other.
I added ssDNA and dsDNA together with enough T4 DNA ligase (NEB), and
ssDNA:dsDNA = 100:1 (I just hope ligation efficiency is high). I
incubated the sample at 16 C for 12 hour and then 65 C for 20 min to
inactive T4 ligase.
From the 1% agarose gel, I can get ~2.1-kbp band which means the ssDNA
+ dsDNA. However, when I used gel extraction kit (QIAGEN) to cut the
band out and purify it again, and run another gel to check it, this
~2.1-kbp band lost. I guess ~2.1-kbp band I got just an annealing
product (15-nt overhang is long enough to anneal each other, right?),
but the ligation did not work. It is because my dsDNA is too long
(~2-kbp) to block the T4 ligase to bind on the ligase position?
Another thing I know is that the yield of single strand ligation is
lower than the normal ligation (two strand ligation)
I will be grateful if you can give me some idea and suggestion.
Waiting for your reply.
Best Regards,
Zhi Qi
Biophysics Department
UIUC
