ligation problem.. - (Jul/24/2005 )
hi all...
i did ligation of 18 kb vector to 4.8 kb insert and transform it. but unfortunately untill now i still didnt get any colonies at all..the ratio vector to insert was 1:8. and i wonder do we need to inactivate out ligase before transformation?is it better for to inactivate ligase before transform it?some people told me better not to..im confuse..
i did ligation of 18 kb vector to 4.8 kb insert and transform it. but unfortunately untill now i still didnt get any colonies at all..the ratio vector to insert was 1:8. and i wonder do we need to inactivate out ligase before transformation?is it better for to inactivate ligase before transform it?some people told me better not to..im confuse..
It is advised to inhibit T4 DNA Ligase b4 transformation which o'wise hinders transformation efficiency. I do it for my NEB ligase by heating at 65C for 10 min.
How did you prepare your insert?
PCR and then cut it, and if so how many extra basepairs did you have left besides your restriction site (you will need more than just the recommended 6)?
Did you gel-purify any of it (possible UV-damage)?
I've done transformation with both inactivated and non-inactivated ligase, it might make a difference, but if your ligation is efficiënt enough you should be able to get colonies.
PCR and then cut it, and if so how many extra basepairs did you have left besides your restriction site (you will need more than just the recommended 6)?
Did you gel-purify any of it (possible UV-damage)?
I've done transformation with both inactivated and non-inactivated ligase, it might make a difference, but if your ligation is efficiënt enough you should be able to get colonies.
the insert was digested with hindIII . sorry for the late reply
