Kun:LabNotes/ExonomeSeq/2007-12-21
Exp. 1 Perform another round of E55kv1 capturing experiment
Shearing template DNA
10ul 353ng/ul GM10835 gDNA + 750ul shearing buffer (1X TE w/ 10% Glycerol) -> sheared with Nebulizer with unknown pressure for 1min -> recovered 650ul -> isopropanol precipitation with the presence of 4ul linear acrylamide.
Resuspended in 10ul ddH2O -> ~300ng/ul
Circularization reaction
Sheared NA10835(~300ng/ul) 3ul E55kv1 probes 107nM 17ul 10X Ampligase buffer 2.25ul
95C 10min -> 60C 40h -> add 2.5ul NSL mix -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold
Colony PCR
Yesterday I pooled the transformations for 180/280bp and 470bp fragment in one plate, and then 150bp and 350bp fragments in another plate. I have only <10 colonies in the first plate and >200 colonies in the second. Today I decided to pick 8 colonies from each plate for PCR.
X 17 2X Taq master mix: 15ul 255ul 10uM M13 F/R mix: 1ul 17ul H2O 14ul 238ul
94C 3min -> (94C 30sec -> 55C 30sec -> 72C 1min) x 35 -> 72C 3min -> 4C hold.