adapter liagation into a single cut plasmid? - (Jul/25/2007 )
Before I really do it, I need help.
I want to ligate two adapters to BOTH ends of a single Ecor I cut plasmid. How to do it ?
I will synthesize two adapers , then annealing into complementary double strands adapters with Ecor I stricky ends.Incubate with a single Ecor I cut plasmid at the presence of T4 ligase overnight ? Does it work? My question is what is the ration between self ligation of single cut plasmid and adapter ligation with Ecor I sticky ends?
Will the self ligation of Ecor I single cut plasmid dominant after ligation? I prefer to get dominant adapters ligation with both ends of the Ecor I single cut plasmid.
AFAIK you have to use the adapters in huge excess. Then you'll get less self-ligation. Of course you'd get concatamers of the adapter, but then you can use your other RE (not EcoRI) to eat it down to only one. Then gel-purify of course. If you want to further improve the efficiency, maybe you want to make your vector blunt-ended, and do the whole stuff like that. You have to take care that you don't have recognition site for your other RE in the vector somewhere else.
i did that experience 2 times...
Well, first is to properly dephosphorylate the vector and properly phosphorylate your oligos (or if you can afford, order them phosphorylated, but home made is reliable).
second, first ssots should be done with 1:5 and 1:10 ratio.
If you face self religation, increase the amount of adaptater.
in case of several concatemers, i would not gel purify and religate. The 2 plasmids should be 100bp different, and it's quite hard to separate 6 to 6.1 kpb isn't it ?
depending of the length of the fragment you want to remove, an artificial raise in the cut off of a column can be done (as mentionned by the nucleospin II manual of Macherey nagel.
thanks a lot.
I just synthesize two oligos , then make them annealed into double strands adapter. Only one end of this adapter is complementary with Ecor I cut site. Another end of adapter can not be complementary with Ecor I and each other. I just want to ligate this adapeter with a single cut plasmid/Ecor I in both ends of the linerized plasmid. So it is impossilbe to get concatermers , right? Theoritically, only one copy of adapter can liagate with one end of linearized plasmid and another adapter with another end of the plasmid , right ? I just worry that the both ends of linearized plasmid will not ligate with the ends of the adapter dominantly but self ligate.
Sorry ,English is my weakness.
I am digesting a Topo construct with BamHI/Sal HF and cloning into another vector cut with Bam Hi/ HindIII and using the corresponding adapters in simultaneous ligation of all the fragments ; but not experiencing any success even after several attempts.
1.I used NEB quick ligase buffer with regular T4 DNA ligase for 15mts as well as overnight ligation.
2. used electroporation (clone size 11kb) and chemical transformation.
Thankscloning with adapters