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='''Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)'''= ==Objective== *Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons. ==Materials== *Enzyme - Taq 2X master mix (New England Biolabs) *Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL *Human genome primers **18S-306 (cho. 21) - previously tested **2-1 (cho.2, 540 bp, Tm 52C) **3-1 (cho.3, 235 bp, Tm 52C) *Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O ==Exp. Design== Templates (A->H) --------------------------------------------------------------------------- A3 A6 A7 B4 B7 C3 C4 C8 p-Pos p-Neg Pos H2O --------------------------------------------------------------------------- Primer Strip1 P1 (18S-306) stirp2 P2 (2-1) strip3 P3 (3-1) ==Procedures== *Thaw the Taq2X enzyme and primers. *Set up 52C program. *Preppare 5 trips of 8-well PCR tubes and transfer the 1 uL template. *Master Mix - x 5 tubes: 1 rxn 14 rxn ---------------------- H2O 3.5 49.0 Primer 0.5 7.0 - primer is differnt in each one of three master mix Taq 2X 5.0 70.0 ---------------------- 9.0 126.0 (uL) 126/14=9 *Transfer 9 uL of master mix into each reaction *Perform PCR reaction **Strip 1 goes to block A (program GENE59: Tm59, 30 cycles) **Strip 2-3 go to block B (program GENE52: Tm52, 30 cycles) *Gel Electrophoresis **1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb) **Run at 135 V for 20 min. ==Results== '''FIG. Gel electrophoresis picture ''' [[Image:ZhangLab_2 2009-02-26 test2 primer_18S.bmp|600px]] [[Image:low mass ladder.bmp|150px]] '''Test by primer 18S (306 bp)''' [[Image:ZhangLab_2 2009-02-26 test2 primer_2-1.bmp|600px]] '''Test by primer 2-1 (540 bp)''' [[Image:ZhangLab_2 2009-02-26 test2 primer_3-1.bmp|400px]] '''Test by primer 3-1 (235 bp)''' ==Result & Discussion== *Samle B7 and p-Pos contains positive bands by 18S primer (Cho.21/Un) and primer 3-1 (Cho.3). *A very weak band was also found (but not showing clearly in pic) on B7 by primer 2-1 (Cho.2). However the p-Pos failed to show it. **Sample p-Pos should contain successful amplification. The faliure of p-Pos may be caused by the interference of junk (salt?) left in MDA PCR product. **Clean the junk using PCR product purification kit and test the PCR again on those p-Pos sample.
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