A speical ligation case ( a strange ssDNA to dsDNA)
Posted 15 February 2011 - 12:18 AM
Such DNA construct is composed of short ssDNA(30 bases) sandwiched between two long dsDNA(~600bp each);
It looks like this: (not in scale)
Right now I have 2 samples:
(1) The 5'-handle dsDNA, with a 4 bases 5'-overhang (-TGGG-5') on the right-hand side. (Black portion)
(2) The ssDNA that is already attached to the 3'-handle. The phosphorylated 5'-end of the ssDNA terminates with 5'-ACCC. (Red portion)
Both DNA have their own labeling at one end (Biotin or Digoxgenin)
And I want to use T4 DNA ligase (NEB) to ligate them into my final construct.
Does anyone know that such weird "dsDNA+ partial ssDNA" ligation will work or not?
The last time I tried this ligation, I get only very, very weak band near the predicted position (1.3K)..... cannot be sure whether it is the real product or not.
The protocol I used is:
5'-handle 6.97uL (contains 0.85 pmole or 350ng DNA)
3'-handle 2.7uL (contains 0.85 pmole or 377ng DNA)
10X T4 DNA ligase buffer(NEB) 2 uL
T4 DNA ligase(NEB) 1uL (contains 400 cohesive units)
Keep in 16oC for 24 hr (no product if less than 12 hr), then immediately resolve in 2% agarose gel.
What I worry the most is that such "sticky end" is only 4 bp long.... would it be a problem?
Or should I use other type of ligase? I really want to improve the ligation efficiency.
I'll be grateful if you can help me about this.... get stocked for a couple of month already
Posted 15 February 2011 - 02:43 PM
Posted 17 February 2011 - 03:34 AM
Thanks very much for your reply
Talking about T4 RNA ligase, I've tried it before but sadly it doesn't work... Adding an oligo to make it ds seems a good idea. I'm planning to order such an "helper" oligo (~20bp) to aid my ligation, after that I'll add another oligo that complementary to the helper, in excess amount, to compete and take away the helper oligo as much as possible during denaturation-recovery. (There will be a fluorescein labelled on the 5'-end of the helper oligo to tell me the efficiency of such removal.) How do you think about this method??
Posted 17 February 2011 - 04:49 AM
Posted 18 February 2011 - 05:34 AM
Thanks for these advices, if the current methods don't work, I'll try them.