useing a uniqeu enzyme for both insert and vector digestion - (Oct/05/2011 )

Dear Phage 434
thank you indeed for the elaborated explanation, may I ask what method do you use to prepare chemically competent Ecoli? the standard CaCl2 method? I use MOPS buffer and compared to the CaCl2, looks like it works better.

http://openwetware.org/wiki/TOP10_chemically_competent_cells

Thank you indeed, I will try it out. just another question, any suggestions for increasing the ligation efficiency, specially when using the same enzyme for both vector and Insert?

Ligation efficiency shouldn't go down just because you are using the same enzyme for both sides of your insert/vector. The efficiency is dependent on the type of overhang, the phosphorylation status of the vector/insert, the size of the vector/insert, and the ratio of vector:insert. SalI has a 4-base 5' overhang which is ideal for ligations. The only problem you will have is orientation, since your insert can go into the vector both backwards and forwards, but you just have to screen for orientation by restriction digest or PCR after you miniprep. You have to phosphatase your vector to prevent self-ligation, which you have done, and that can also decrease your ligation efficiency, but not so much that you should have a problem.

Lastly, I will strongly recommend that you stop screening with colony PCR and start mini-prepping plasmid and screening that way. Colony PCR is a great quick and dirty way to screen clones when things are working, but once you start having so many problems, you want to be using samples that you can depend on. This includes using millipore water (or PCR water if you buy it). There can be all kinds of enzyme inhibitors present in crude DNA preps, colony PCRs, and non-standard water sources, and right now, you need to eliminate as many questions as possible.

Best of Luck.

hello all

thank you for the support and tips, IT FINALLY WORKED.
hooray