Sam:LabNotes/Microbione/2009-3-2: Difference between revisions
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>Sam Chiang (New page: =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 exi...) |
>Sam Chiang No edit summary |
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**Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns) | **Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns) | ||
**Set2: 1-2 ~ 22-2 and X-2 (23 rxns) | **Set2: 1-2 ~ 22-2 and X-2 (23 rxns) | ||
==Procedures== | ==Procedures== | ||
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**Total reactions: 12 rxns | **Total reactions: 12 rxns | ||
1 rxn | 1 rxn 24+3 rxn | ||
--------------------------- | --------------------------- | ||
H2O 3.5 | H2O 3.5 94.5 | ||
Template 1.0 27.0 | |||
Taq 2X 5.0 | Taq 2X 5.0 135.0 | ||
--------------------------- | --------------------------- | ||
9. | 9.5 256.5 (uL) 256.5/27=9.5 | ||
*Transfer | *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 | |||
*Transfer | *Perform PCR reaction in thermocycler: program GENE52 (Tm52, 30 cycles) | ||
*Perform PCR reaction in thermocycler | |||
*Gel Electrophoresis | *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 | **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 | **Run at 135 V for 18 min. | ||
**Loading: Sample 7 uL + 2 uL | **Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL | ||
**Loading order (Left to right): | **Loading order (Left to right): 1-1 -> 22-1 -> X-1 ->Y-1 -> Low mass ladder | ||
*Repeat the same design to perform PCR on primer set2 (23 reactions) | |||
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[[Image:ZhangLab_2 2009- | [[Image:ZhangLab_2 2009-03-03 15hr 01min-set1.jpg|800px]] | ||
[[Image:ZhangLab_2 2009- | [[Image:ZhangLab_2 2009-03-03 15hr 01min-set2.jpg|800px]] | ||
*Upper: Test by primer 18S (306 bp) | *Upper: Test by primer 18S (306 bp) | ||
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==Discussion== | ==Discussion== | ||
Revision as of 07:30, 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
- Loading order (Left to right): 1-1 -> 22-1 -> X-1 ->Y-1 -> Low mass ladder
- Repeat the same design to perform PCR on primer set2 (23 reactions)
Results
File:ZhangLab 2 2009-03-03 15hr 01min-set1.jpg
File:ZhangLab 2 2009-03-03 15hr 01min-set2.jpg
- Upper: Test by primer 18S (306 bp)
- Lower: Test by primer 2-1 (540 bp)