MAD confirmation for test 3
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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 (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 (1 uL): 1/100 diluted MDA products
- A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1 ng/uL gDNA), H2O
Templates (A->H) --------------------------------------------------------------------------- A3 A6 A7 B4 B7 C3 C4 C8 p-Pos p-Neg Pos H2O --------------------------------------------------------------------------- Primer P1 18S-306 (cho. 21, 306 pb, Tm 59C) P2 2-1 (cho.2, 540 bp, Tm 52C) P3 3-1 (cho.3, 235 bp, Tm 52C)
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 3 (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
- Prepare 3 trips of 8-well PCR tubes and transfer the 1 uL template.
- Transfer 9 uL of master mix into each reaction
- Perform PCR reaction in thermocycler
- Strip P1 goes to block A (program GENE59: Tm59, 30 cycles)
- Strip P2 and P3 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.
- Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
Results
File:ZhangLab 2 2009-02-28 test2 primer 18S.bmp File:ZhangLab 2 2009-02-28 test2 primer 2-1.bmp
- Left: Test by primer 18S (306 bp)
- Right: Test by primer 2-1 (540 bp)
Result & Discussion
- 1/100 dilution didn't give us better PCR amplification. Even the p-Pos didn't show up.
- The PCR template using MDA amplicon might be used in a range between 1/1 ~ 1/10 dilution.