CLONING LONG INSERT RATIO INS:VEC - (Feb/29/2008 )
hI!
i'M TRYING TO CLONE AN INSERT OF 6KB INTO A PLASMID 5.1KB. THE INSERT IS A PCR PRODUCT KINASED AND THE VECTOR IS DESPHO.
THE NEG CONTROL IS OK. (AUTOLIGATED VECTOR). THE POSITIVE CONTROL IS OK TOO. (VECTOR PLUS ANOTHER INSERT).
I' TRIED RATIO INS:VECTOR 1/3, 1/4, 1/5. bUT ONLY 3 OR 4 COLONIES GROW AND THEY DO NOT HAD THE INSERT.
WHAT DO YOU SUGGEST?
tHANK YOU VERY MUCH FOR YOUR ANSWER!!
I' M CRAZY! WITH THIS EXPERIMENT I FINALLY MY THESIS!!
The correct ratio is molar 1:1. In this case, this is almost 1:1 on a weight basis too. Think about it -- the ligase has no idea whether the DNA it is ligating is a vector or not. You may also be using too high a concentration of DNA. 10-20 ng of each in a 10 ul volume is enough. This sounds like a blunt ligation -- how are you doing it?
Always I did it with 100ng of the vector, 20 ul of final volumen, transform with "all the ligation volumen" with 50ul of competents cells and I had success (but with cohesive blunts).
I have read in this forum that the ligation volumen could be toxic. but until now i had not problems.
I have tried 1:1 and 2:1 in this weekend and today i will try the transforming experiment. I 'll see
Thank you !!
sometimes we have used very high amounts of DNA (100-200ngs-total) in ligations and they have worked.
Are you digesting the vector/insert with any enzyme?
Are you digesting the vector/insert with any enzyme?
The vector was treated with EcoRV (blunt ends)/ CIAP and the insert with klenow PLUS T4 KINASE. The controls are ok (vector autoligated and the vector plus another insert).
thank you