Kun:LabNotes/ASE/2007-12-5

From ZhangLabWiki
Jump to navigation Jump to search

Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007[edit]

                              one reaction        3 reactions
          Template:              5ul                  each
          10x Buffer:          9.5ul                28.5ul
          25mM MgCl2             6ul                  18ul
          25mM dNTP            1.2ul                 3.6ul
          100uM AmpSolV6F2     0.4ul                 1.2ul
          100uM AmpSolV6R2     0.4ul                 1.2ul
          Platinium Taq          1ul                   3ul
          50X SYBG I           0.8ul                 2.4ul
          H2O                 76.7ul                 231ul

94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.

   File:2007-12-05-cDNA-CES22k-PCR-PAGE.png

Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07[edit]

Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency.

Reference: H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM.
Template:

  1. 12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each
  2. 11-26-07: BJ cDNA 1ul
  3. 11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each

Two 50ul reactions for each template.

                              one reaction        24 reactions
          Template:              1ul                  each
          10x Buffer:            5ul                 120ul
          25mM MgCl2             3ul                  72ul
          25mM dNTP            0.6ul                14.4ul
          100uM AmpV6F         0.2ul                 4.8ul
          100uM AmpV6R         0.2ul                 4.8ul
          iTaq                 0.5ul                  12ul
          50X SYBG I           0.4ul                 9.6ul
          H2O                   39ul                 936ul

94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold.

Well / Set Sample C(t) pM*ul # molecules/ul
A2 H1(1) 10.43 0.033 19530
B2 H1(1) 10.11 0.041 24888
C2 H2(1) 10.55 0.030 17790
D2 H2(1) 10.31 0.035 21276
E2 Hues6(1)? 7.91 0.220 131760
F2 Hues6(1)? 8.01 0.204 122580
G2 BJ(1)? 8 0.206 123660
A3 H1(2) 12.49 0.007 4075.8
B3 H1(2) 12.03 0.010 5799.6
C3 H2(2) 11.15 0.019 11262
D3 H2(2) 12.08 0.009 5569.8
E3 Hues6(2) 8.73 0.118 71040
F3 Hues6(2) 8.97 0.098 59052
G3 BJ(2) 12.75 0.006 3358.2
H3 BJ(2) 12.28 0.008 4779

I'm not completely sure whether I added the right template to E2,F2,G2,H2.