6 replies to this topic

[Dott. Berrino]

I need to ligate together two single fragments of dsDNA obtained from two digestion reactions with the same restriction endonuclease which generates sticky ends.  I also need to obtain a clear band on an agarose gel for my cloning strategy. I've tried several times with T4 DNA ligase but I couldn't obtain the desired band. (I obtain no band or a smear)
Is there anyone who can help me?

[wirly]

We need more info.  What is the restriction enzyme?  How are you digesting?  How are you purifying the fragments?  What are the details of  the ligation reacion you are doing?

[fred_33]

hi
what are the respective sizes of your fragments?

[Dott. Berrino]

Hi
I cut the two fragments with Bcl I (cuts just once in each fragment leaving cohesive ends). The digestion was performed 1 hr at 50 °C and inactivated 15 min at 65 °C. I obtained two fragments: size 691 and 583 bp.
I couldn't purify my fragments after digestion: do you think is absolutely necessary? In fact the other two bands generated after digestion are so small around 30bp that I suppose they are lost.
I tried ligation with different concentration of Dna:
Dna from 0,1  to 1 micrograms.
Enzyme 0,1 U  for 1 hr at 25 °C (protocol for cohesive ends). I'm expecting a band of 1274 bp. How do you suggest to proceed? Thank you very much for your help!
ciao

[fred_33]

hi

i was wondring, how did you otained your fragments? Do you mean they are with blunt ends both before the digestion? Is there a possibility that the extremities of these fragments show partial complementarity?

for your ligation reaction, i obtained more efficiency by ligation overnight at 16°. I also have used the quick ligase kit from neb with good results.
For the proportions of your two fragments, i would recommend you to do three assays ; first  with an equivalent mass of each fragmentn, and for the two others, consider integrating a fragment in the other. That says for the assay "2" you put 1x mass of 691pb (50ng) and 5x mass 583pb (210ng), and for the assay "3",  you put 1x mass of 583pb(50ng) and 5x mass 691pb(296ng).
One of the three should work correctly.

Fred

[Dott. Berrino]

Ok that's a good idea! I'll try and I'll let you know. Anyway my fragments have both blunt ends  before digestion and should have cohesive ends at one side after digestion.So my problem should really be due to  ligation. I didn't try O.N. at 16 °C  because it was suggested as the ideal protocol for blunt ends ligation and I wanted to avoid this. Thank you again for your help
ciao

[Dott. Berrino]

You were right fred!
I tried different conditions as you suggested and in one case it seems to work!!
Now I will sequence my product to confirm it's the right one.
thanks again,
au revoir from Italy