Kun:LabNotes/ExonomeSeq/2007-12-21

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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.