4 replies to this topic

[F.Dynasty]

Hi everyone, I'm working on a project utilizing optical tweezers to "stretch"  my DNA constructs in order to get mechanical information.
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:
DDW  8.33uL
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)                  
  ---------------------------------------------------
                                                                                                 Total: 20uL    
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

[phage434]

These ligations basically don't work.  Ligase really needs to see doubled stranded DNA on both sides of its ligation site, and 4 bp is not enough.  You could try switching to T4 RNA ligase, which might ligate under these circumstances, since it is pretty promiscuous.  Adding an oligo to make the ss DNA fragment ds during ligation would work, but you'd need a good way to remove it.  I would have suggested a biotin label and strep coated beads, but you already are using a biotin.  Not sure what to suggest.  Perhaps you could use a nicking enzyme to cut a fully ds strand on one side, then denature in dilute NaOH, recovering just the labeled molecule.  Or anneal a complementary oligo (unlabeled), ligate, then denature and recover the target.

[F.Dynasty]

phage434, on 15 February 2011 - 02:43 PM, said:

These ligations basically don't work.  Ligase really needs to see doubled stranded DNA on both sides of its ligation site, and 4 bp is not enough.  You could try switching to T4 RNA ligase, which might ligate under these circumstances, since it is pretty promiscuous.  Adding an oligo to make the ss DNA fragment ds during ligation would work, but you'd need a good way to remove it.  I would have suggested a biotin label and strep coated beads, but you already are using a biotin.  Not sure what to suggest.  Perhaps you could use a nicking enzyme to cut a fully ds strand on one side, then denature in dilute NaOH, recovering just the labeled molecule.  Or anneal a complementary oligo (unlabeled), ligate, then denature and recover the target.

To phage434,

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??

F.Dynasty

[phage434]

Depending on how long your regions are, you could also simply order the upper fragment as a single oligo (or use asymmetric PCR with a biotin primer to make it).  Then simply anneal the two other fragments.

[F.Dynasty]

phage434, on 17 February 2011 - 04:49 AM, said:

Depending on how long your regions are, you could also simply order the upper fragment as a single oligo (or use asymmetric PCR with a biotin primer to make it).  Then simply anneal the two other fragments.

Thanks for these advices, if the current methods don't work, I'll try them.