Sam:LabNotes/Microbione/2009-3-2
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Human genome screening primer set - quality validation
Objective
- Human whole genome primer sets were obtained from Professor (2 sets).
- The primer set were used to confirm the existance of choromosomes in MDA amplicon using multiplex PCR.
- Individual validation of each primer is required before performing multiplex PCR.
Samples & Materials
- Enzyme - Taq 2X master mix (NEB)
- Templates (1 uL): 1 ng/uL human gDNA (NEB)
- Primers: All primers are diluted to 100 uM, and mixed basd on the gene (Forward 20 uL + Reverse 20 uL + H2O 160 uL)
- Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
- Set2: 1-2 ~ 22-2 and X-2 (23 rxns)
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
File:ZhangLab 2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp
File:ZhangLab 2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp
- 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