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=Human cell MDA confirmation for test3= ==Objective== *PCR test on the 10 candidates MDA amplicons picked up from Test 3. ==Samples & Materials== *Enzyme - Taq 2X master mix (NEB) *Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution *Primers: 18S-306 (cho. 21, 306 bp, Tm 59C; 2-1 (cho.2, 540 bp, Tm 52C) ==Exp. Design== Templates (A->H) ----------------------------------------------------------- A2 A4 A5 A7 B1 B2 B4 B6 B7 C7 p-Pos p-Neg ----------------------------------------------------------- Primer P1 18S P2 2-1 Templates were tested in original and 1/10 dilution ==Procedures== *Thaw the Taq2X enzyme and primers. *Dilute all MDA template. Prepare diluted Pos template (1 ng/uL) *Set up PCR program (GENE59, GENE52). *Master Mix - x 2 (Primers are various) **Total reactions: 12 rxns 1 rxn 12+2 rxn --------------------------- H2O 3.5 49.0 Primer 0.5 7.0 Taq 2X 5.0 70.0 --------------------------- 9.0 126.0 (uL) 126/14=9 *Transfer the 1 uL template to PCR tubes (8-well strip) *Transfer 9 uL of master mix into each reaction *Perform PCR reaction in thermocycler **Strip P1 goes to program GENE59 (Tm59, 30 cycles) **Strip P2 goes to program GENE52 (Tm52, 30 cycles) *Gel Electrophoresis **1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 2 midium tray with 26-well comb **Run at 135 V for 20 min. **Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL **Loading order (Left to right): A2->P-Neg (original dilution) -> laddr -> A2->P-Neg (1/10 dilution) ==Results== [[Image:ZhangLab_2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp|800px]] [[Image:ZhangLab_2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp|800px]] *Upper: Test by primer 18S (306 bp) *Lower: Test by primer 2-1 (540 bp) ==Discussion== *Even 1/10 dilutio didn't help improve the regular PCR performance a lot. The p-Pos results still looks very weak in prier 2-1 test *Pick up amplicons from B1 and B4 for 2nd MDA amplification
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