Sam:LabNotes/Microbione/2009-3-2: Difference between revisions
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**Run at 135 V for 18 min. | **Run at 135 V for 18 min. | ||
**Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL | **Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL | ||
*Repeat the same design to perform PCR on primer set2 (23 reactions) | *Repeat the same design to perform PCR on primer set2 (23 reactions) |
Revision as of 07:47, 5 March 2009
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)
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 24+3 rxn --------------------------- H2O 3.5 94.5 Template 1.0 27.0 Taq 2X 5.0 135.0 --------------------------- 9.5 256.5 (uL) 256.5/27=9.5
- Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip)
- Transfer the 0.5 uL primer into each reaction
- Perform PCR reaction in thermocycler: 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 18 min.
- Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL
- Repeat the same design to perform PCR on primer set2 (23 reactions)
Results
File:ZhangLab 2 2009-03-03 15hr 01min-set1-new.jpg
File:ZhangLab 2 2009-03-03 15hr 01min-set2.jpg
- Upper: Test by primer set1 (chromosome 1 ->8, ladder, 9->16, ladder, 17-22, X, Y)
- Lower: Test by primer set2 (chromosome 1 ->8, ladder, 9->16, ladder, 17-22, X)