No luck with blunt and sticky ligations, need hlep - Prepare for a thorough read (Aug/24/2010 )
Ok so I now have a protocol that at least yields more colonies than normal. I'd like to share this:
Blunt fragments that have 1 or 2 side 3' ends with T4 polymerase.
Blunt fragments that have 2 side 5'with Klenow LG
After digestion and Klenow, column purify the vector fragments. Sap 1h @ 37C with AP. Then bead-purify the blunted, DP vectors. Bead purify the insert too. Wait until the pellet of beads looks like power sugar. Solve pellet in 25ul 10mM TRIS (pH 8.5) 10'@50C. Spin down, collect sup. Add 25ul to the pellet and solve again. Combine sups and spin down 2x @ 13000 each time transferring the sup to a fresh tube.
Ligate 50ng vector, F:V = 10:1 (mol:mol), total volume of ligation 25ul, 100U T4 ligase, 10% PEG. Incubate O/N @ 16C
We have lousy bacterias so I transform 16 (sixteen) ul of my ligation mix to 50ul of these bacteria and it gives 4-10 times more colonies than 2ul.
The j/i change works almost as well as PEG. 1mM HCC I couldn't get to work.
But now I have this other problem:
I can't seem to get any clones with my blunted ClaI digested vector?
Why must this be? ClaI only has 2bp 5' overhangs. Could 15' @ 25C with Klenow LG be too much? Could it be that when it's finished after two nucleotides it gets bored and adds more bp's so it produces a 3' overhang (and can't chew that away because its 3' exonuclease activity is low)?