Kun:LabNotes/ASE/2007-12-30: Difference between revisions

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==Exp.2 Perform QPCR to test whether the PGP1 cDNAs contain gDNA contaminations==
==Exp.2 Perform QPCR to test whether the PGP1 cDNAs contain gDNA contaminations==
To estimate the level of gDNA contaminations, I use two sets of six PCR primer pairs (A1,B1,C1,F1,G1,H1) in the PGP1VER2 plates, one set targeting cDNA and the other targeting gDNA.   
To estimate the level of gDNA contaminations, I use two sets of six PCR primer pairs (A1,B1,C1,F1,G1,H1) in the PGP1VER2 plates, one set targeting cDNA and the other targeting gDNA.   
                              GM20431   GC1F     GC1EP     Hues6    x 50
                      GM20431(06/05) GC1F(06/05) GC1EP(06/08) Hues6(05/22)     x 50
          Template(2ng/ul)      1ul       1ul     1ul       1ul      12 x 4
      Template(2ng/ul)      1ul         1ul         1ul         1ul      12 x 4
          2X Taq Master mix    15ul     15ul     15ul     15ul      750
      2X Taq Master mix    15ul         15ul       15ul         15ul      750
          2uM primer mix        5ul       5ul     5ul       5ul      each
      2uM primer mix        5ul         5ul         5ul         5ul      each
          50X SYBG I          0.2ul     0.2ul   0.2ul     0.2ul      10
      50X SYBG I          0.2ul       0.2ul       0.2ul       0.2ul      10
          H2O                8.8ul     8.8ul   8.8ul     8.8ul      440
      H2O                8.8ul       8.8ul       8.8ul       8.8ul      440


94C 2min -> (94C 30sec -> 57C 30sec -> 72C 45sec)x 40 -> 72C 3min -> 15C hold
94C 2min -> (94C 30sec -> 57C 30sec -> 72C 45sec)x 40 -> 72C 3min -> 15C hold

Revision as of 01:16, 31 December 2007

Exp.1 Probe prep. for the CES22k set on two new libraries

PCR (with iTaq)

                             x1        x16x2 
        template(100nM)     0.1ul     2x1.6ul    
        10x ABI buffer       10ul       32ul
        25mM MgCl2            6ul       192ul
        10mM dNTP             2ul        64ul
        100uM pAP1V6U       0.3ul       9.6ul
        100uM phosAP2V6     0.3ul       9.6ul
        50X SYBG I          0.8ul      25.6ul
        iTaq                  1ul        32ul
        H2O                  80ul      2560ul

94C 3min -> 16 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Purify each amplicons with 2 Bioneer columns.

Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 4h, 75C 15min, Purify each amplicons with 2 Bioneer columns, eluted in 100 elution buffer.

Dpn II & USER digestion: Add 20ul 10X Dpn II buffer, 10ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 8h, 75C 10min.

Exp.2 Perform QPCR to test whether the PGP1 cDNAs contain gDNA contaminations

To estimate the level of gDNA contaminations, I use two sets of six PCR primer pairs (A1,B1,C1,F1,G1,H1) in the PGP1VER2 plates, one set targeting cDNA and the other targeting gDNA.

                      GM20431(06/05) GC1F(06/05) GC1EP(06/08) Hues6(05/22)     x 50
     Template(2ng/ul)      1ul          1ul         1ul          1ul       12 x 4
     2X Taq Master mix    15ul         15ul        15ul         15ul       750
     2uM primer mix        5ul          5ul         5ul          5ul       each
     50X SYBG I          0.2ul        0.2ul       0.2ul        0.2ul       10
     H2O                 8.8ul        8.8ul       8.8ul        8.8ul       440

94C 2min -> (94C 30sec -> 57C 30sec -> 72C 45sec)x 40 -> 72C 3min -> 15C hold