Sam:LabNotes/Microbione/2009-3-3
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Human genome screening primer set - quality validation[edit]
Objective[edit]
- Human whole genome primer sets were obtained from Professor (2 sets).
- The primer set were used to confirm the existence of chromosomes in MDA amplicon using multiplex PCR.
- Individual validation of each primer is required before performing multiplex PCR.
Samples & Materials[edit]
- 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[edit]
- 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 medium 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[edit]
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 (1-1 -> 8-1, ladder, 9-1 -> 16-1, ladder, 17-1 -> 22-1, X-1, Y-1)
- Lower: Test by primer set2 (1-2 -> 8-2, ladder, 9-2 -> 16-2, ladder, 17-2 -> 22-2, X-2)
- Primer name (amplicon size)
1-1 (270) 4-1 (383) 7-1 (242) 10-2 (299) 13-2 (1102) 16-2 (1324) 19-1 (1007) 22-2 (1166) 1-2 (676) 4-2 (896) 7-2 (498) 10-1 (214) 13-1 (679) 16-1 (806) 19-2 (1512) 22-1 (757) 2-1 (540) 5-1 (413) 8-2 (231) 11-1 (131) 14-1 (598) 17-2 (1240) 20-1 (174) x-2 (839) 2-2 (649) 5-2 (530) 8-1 (230) 11-2 (525) 14-2 (756) 17-1 (419) 20-2 (584) x-1 (147) 3-1 (235) 6-2 (1050) 9-1 (373) 12-1 (984) 15-1 (297) 18-2 (288) 21-1 (200) y-1 (122) 3-2 (1323) 6-1 (299) 9-2 (907) 12-2 (1689) 15-2 (1422) 18-1 (171) 21-2 (346)
Discussion & Suggestion[edit]
- Except primer set1 chromosome X and Y which have only weak bands, all primers generate strong band with good amplification at 52C
- The PCR cycle is too many -> saturation of amplicons
- The primer concentration used in PCR is too high -> serious primer dimer seen on the gel