blunt end ligation problem - (Mar/21/2007 )
I am doing overexpression expriemnt..
In that i am trying to ligate blunt end insert( 1.4 kb) with blunt end vector(11kb) which is made blunt ended by maker itself.
but unfortunately i dont know i couldnt get any colonies after transformation with dh5 alpha....
please suggest me suitable protocol...or any clue ...
1. Dephosphorylate your vector
2. Phosphorylate your PCR insert by T4 polynucleotide kinase
3. Gel purify both of them.
4. Calculate the amount of PCR fragment you are going to use, based on your vector. i used 30ng of my vector.
5. Use fresh T4 ligase, better to be concentrated. i remember there is some ligase concentrated 2000u/ul.
6. Screen as many colonies as you can. i got 1 out of my 50 mini-prep.
Sometimes I use around 100-200ng of DNA for ligation. Can help if its blunt.
many people use quick ligation kit for difficult blunt end ligations.
good Luck !!!
thanks alot both of u for your valuable guidance
This time transformation i got only two colonies,...
But during ligation i used only 20ng insert DNA and 50 ng vector....so is it ok?..or should i do again ligation with more DNA and vector....
try to analyse the 2 colonies, it could be right. and one needs only a single right colony.
good luck !!!