Sam:LabNotes/Human/2009-2-26
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Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)[edit]
Objective[edit]
- Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons.
Materials[edit]
- 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[edit]
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[edit]
- 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[edit]
FIG. Gel electrophoresis picture
File:ZhangLab 2 2009-02-26 test2 primer 18S.bmp File:Low mass ladder.bmp Test by primer 18S (306 bp)
File:ZhangLab 2 2009-02-26 test2 primer 2-1.bmp Test by primer 2-1 (540 bp)
File:ZhangLab 2 2009-02-26 test2 primer 3-1.bmp Test by primer 3-1 (235 bp)
Result & Discussion[edit]
- 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.