Kun:LabNotes/ASE/2007-12-30
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.