Ligate two restriction fragments of dsDNA
Posted 09 March 2005 - 09:09 AM
Is there anyone who can help me?
Posted 09 March 2005 - 04:24 PM
Posted 10 March 2005 - 02:16 AM
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!
Posted 10 March 2005 - 03:18 AM
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.
Posted 10 March 2005 - 06:07 AM
Posted 14 March 2005 - 08:36 AM
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.
au revoir from Italy