My problem is that I can't seem to get my insert into my vector -- every clone I get on my "dephosphorylated cut vector + insert + ligase" plates have been self-ligations of the vector. At this point I am guessing the problem has to be ligation, since transformation of my controls is working out as expected. I have tried using different aliquots of ligase buffer and enzyme just in case some of them have "turned" but so far no positive clones. Since my insert is so small (annealed 24mers) is there any way I can check my ligation product to see if the insert is incorporated? Could I digest my ligation product with some of the enzymes whose restriction site is located in the insert?
I have tried ligation reactions ranging from 5 microliters to 15 microliters in volume. I have tried either 50 ng or 100 ng of vector... I have tried a vector:insert molar ratio of 1:1, 1:3, 1:6. I have tried using 2X ligation buffer and 10X.
I am at the point where the lab manager wants me to do PCR cloning but my boss likes things done the "digest and ligate" way. I really truly appreciate any help or advice you might have!
Edited by yoursisterdebra, 11 January 2011 - 01:13 PM.