I have a PCR fragment with blunt edges that I'd like to insert into my vector. I already inserted it in a normal forward direction, but now I'd like to try it also in the reverse direction. Does anyone have a suggestion how can I perform the reaction? Thank you very much
Normally a blunted fragment will insert stochastically in both directions, it's just a matter of checking more E. coli clones from your transformation and you should find what you are looking for. It is true that sometimes for unknown reasons even blunted fragments show a preference towards one direction, but I never experienced a 100% preference...
Otherwise you might need to change your cloning strategy.... but for to say anything about this the information is to little in your post!
With blunt ends you will have a mixture of inserts cloned in both directions. If you want to do positional cloning you need to use two different restriction enzymes. Otherwise, follow Pietro suggestion to check more obtained colonies. Good luck
As Pietro said, check more clones for sure you will have some with the inverted fragment!. Don´t forget to choose the correct ER(s) in order to get a different pattern in the gel between the correct and the inverted fragment. Luck!
As mentioned by the others above, with TA cloning this should work! But it means checking more transformants for the direction you're looking for. Another strategy is Gibson Assembly. This will add your insert as you preferred.
Cheers,
Terrens
For blunt-end DNA ligation, the efficiency should be very low. But a modified cloning method might be helpful.:
http://www.sciencedirect.com/science/article/pii/S0003269714000888
You still need to select the possitive one. But anyway, with his this modified method, the selection of possitive clones should be not hard word. Good luck!!
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