MAD confirmation for test 3: Difference between revisions
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>Sam Chiang No edit summary |
>Sam Chiang |
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*Enzyme - Taq 2X master mix (NEB) | *Enzyme - Taq 2X master mix (NEB) | ||
*Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution | *Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution | ||
*Primers | *Primers: 18S-306 (cho. 21, 306 bp, Tm 59C; 2-1 (cho.2, 540 bp, Tm 52C) | ||
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Templates (A->H) | Templates (A->H) | ||
----------------------------------------------------------- | |||
A2 A4 A5 A7 B1 B2 B4 B6 B7 C7 p-Pos p-Neg | |||
----------------------------------------------------------- | |||
Primer P1 18S | Primer P1 18S | ||
P2 2-1 | P2 2-1 | ||
Templates were tested in original and 1/10 dilution | |||
==Procedures== | ==Procedures== | ||
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*Set up PCR program (GENE59, GENE52). | *Set up PCR program (GENE59, GENE52). | ||
*Master Mix - x | *Master Mix - x 2 (Primers are various) | ||
**Total reactions: 12 rxns | **Total reactions: 12 rxns | ||
Line 45: | Line 44: | ||
* | *Transfer the 1 uL template to PCR tubes (8-well strip) | ||
*Transfer 9 uL of master mix into each reaction | *Transfer 9 uL of master mix into each reaction | ||
*Perform PCR reaction in thermocycler | *Perform PCR reaction in thermocycler | ||
**Strip P1 goes to | **Strip P1 goes to program GENE59 (Tm59, 30 cycles) | ||
**Strip P2 | **Strip P2 goes to program GENE52 (Tm52, 30 cycles) | ||
*Gel Electrophoresis | *Gel Electrophoresis | ||
**1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> | **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. | **Run at 135 V for 20 min. | ||
**Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL | **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) | |||
Line 62: | Line 63: | ||
[[Image:ZhangLab_2 2009-02-28 | [[Image:ZhangLab_2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp|800px]] | ||
[[Image:ZhangLab_2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp|800px]] | |||
* | *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 | ||
Latest revision as of 19:59, 2 March 2009
Human cell MDA confirmation for test3[edit]
Objective[edit]
- PCR test on the 10 candidates MDA amplicons picked up from Test 3.
Samples & Materials[edit]
- Enzyme - Taq 2X master mix (NEB)
- Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution
- Primers: 18S-306 (cho. 21, 306 bp, Tm 59C; 2-1 (cho.2, 540 bp, Tm 52C)
Exp. Design[edit]
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[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 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[edit]
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[edit]
- 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