A question about DNA Ligation

[Peter Ellis]

Have you checked that the ssDNA and dsDNA segments have phosphates at 
the appropriate ends?  If you don't have the phosphates in the right 
places, you won't get any ligation.

Peter



Zhi Qi wrote:
> 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
>