Kun:LabNotes/ASE/2007-12-30

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

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 ethanol precipitation, resuspended in 150ul ddH2O.

Lambda exo digestion: to 100ul DNA, add 15ul 10X lambda exo buffer, 5ul lambda exo, 30ul H2O, 37C 4h, 75C 15min, Purify each amplicons with 2 Bioneer columns, eluted in 100 elution buffer. [I lost all DNA with the Bioneer column purification.

Repeat the lamda exo digestion on the remaining 50ul PCR amplicon. 50ul DNA + 6ul 10X buffer + 4ul lambda exo -> 37C 2h -> 85C 10min. Cleanup with G25 column.

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

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

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

 File:2007-12-31-gDNA-contamination-check.png

I think all the PGP1 RNA samples (GM20431, GC1F, GC1EP) have various level of gDNA contamination. Hues6 seem to be cleaner.