A question about DNA Ligation

[Zhi Qi]

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