Kun:LabNotes/ASE/2007-12-5: Difference between revisions

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(New page: ==Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007== one reaction 3 reactions Template: 5ul ...)
 
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94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
==Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07==
Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency.
<b>Reference:</b> H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM. <br>
<b>Template:</b>
#12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each
#11-26-07: BJ cDNA 1ul
#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.

Revision as of 19:02, 5 December 2007

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

                              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.

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

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.