Sam:LabNotes/Microbione/2009-2-25
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Testing of putative amplification from human cell lysate MDA on 02-24-09
Objective
- Using regular PCR (Taq enzyme 2X) to confirm four 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
- 1-1 (cho.1, 270 bp, Tm 52C)
- 2-1 (cho.2, 540 bp, Tm 52C)
- 3-1 (cho.3, 235 bp, Tm 52C)
- 4-1 (cho.4, 383 bp, Tm 52C)
- 18S-306 (cho. 21) - previously tested
- Templates (1 uL): B2, B4, B5, C3, E3(p-Neg), Pos(1/100 gDNA), H2O
Exp. Design
Templates (A->H) ------------------------------------------------------- B2 B4 B5 C4 p-Neg Pos H2O ------------------------------------------------------- Primer Strip1 P1 (1-1) stirp2 P2 (2-1) strip3 P3 (3-1) strip4 P4 (4-1) strip5 P5 (18S-306)
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 8 rxn ---------------------- H2O 3.5 28.0 Primer 0.5 4.0 - primer is differnt in each one of five master mix Taq 2X 5.0 40.0 ---------------------- 9.0 72.0 (uL) 72/8=9
- Transfer 9 uL of master mix into each reaction
- Perform PCR reaction
- Strip 1-4 go to block A (program GENE52: Tm52, 30 cycles)
- Strip 5 goes to block B (program GENE59: Tm59, 30 cycles)