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Stopping sticky ends reannealing?
When I use a restriction endonuclease to cut a plasmid and create sticky ends, how do I stop them reannealing before I can add my gene of interest. Is it just left to probability or is it capped or what??
I really should know this lol Thanks!
I don't mean re-annealing fully, as in ligating together. I mean how do we stop the bases just hydrogen bonding as soon as the enzyme cuts and clears the restriction site. Is it just a matter of probability, i.e. some will re-anneal but some will incorporate the foreign DNA and we just have to find a way to select the one's that have incorporated it.....?
3 Answers
- hcbiochemLv 71 decade agoFavourite answer
For the most part, the sticky ends won't easily anneal at room temperature or at 37C and certainly won't stay annealed permanently. So, you could keep the DNA at 37 or raise the temp briefly to 37 before you add DNA ligase to the reaction.
- 1 decade ago
You could use two different restriction enzymes to cut your plasmid and create sticky ends. You can also add alkaline phosphatase to the vector to prevent reannealing. This removes phosphate groups and stops re-ligation.
- Anonymous1 decade ago
When i make love to my lass, i often wipe my knob on the curtains. They get all sticky for a while, but if i'm tired and i can't be bothered to clean them, i leave them and they go all crusty.
The missus hates it, especially as they are expensive italian curtains her rich nan got her as a house warming gift. She's dead now.
Anyway, what were we talking about?
