Sam:LabNotes/Human/2009-4-12: Difference between revisions
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>Sam Chiang (New page: ='''Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons'''= ==Procedure== *Experiment design Primer 1-1->6-1 Primer 1-1->6-1 Column ...) |
>Sam Chiang No edit summary |
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==Procedure== | ==Procedure== | ||
*Experiment design | *Experiment design | ||
**Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1 | |||
**Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01 | |||
*Thaw | *Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution) | ||
* | *Set up realtime PCR program (decribed as following) | ||
*Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12. | |||
* | *Expemriment arrangements | ||
Column 1 -> 6 Column 7 -> 12 | |||
Primer 1-1->6-1 Primer 1-1->6-1 | |||
B6(3-31) B7(3-31) | |||
D2(3-31) D4(3-31) | |||
D5(3-31) D7(3-31) | |||
D8(3-31) B3(4-01) | |||
B4(4-01) B6(4-01) | |||
C2(4-01) C5(4-01) | |||
C7(4-01) C8(4-01) | |||
POS(100pg) NTC | |||
* | *Prepare diluted primers in PCR tubes | ||
1rxn (8+1)x2 ->20rxns | |||
Primer mix (f+r 10 uM) 1 20 uL | |||
H2O 7 140 uL | |||
-------------------------------------- | |||
* | *Transfer 8 uL diluted primer into each reaction based on the arrangements | ||
* | *Transfer 2 uL diluted templates into each reaction based on arrangements | ||
* | *Prepare master mix in 1.5-mL microcentrifuge tube | ||
1rxn (12+2)x8=112 rxns | |||
Taq (2X) 9.9 1108.8 | |||
* | SYBR (50X) 0.1 11.2 | ||
-------------------------------- | |||
10.0 1120.0 | |||
*Mix well by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor. | |||
*Gently vortex spin down the PCR reaction tubes. Place in Chromo4 machine to perform realtime PCR reaction. | |||
*Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever | |||
Revision as of 01:16, 13 April 2009
Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons
Procedure
- Experiment design
- Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
- Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01
- Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
- Set up realtime PCR program (decribed as following)
- Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
- Expemriment arrangements
Column 1 -> 6 Column 7 -> 12 Primer 1-1->6-1 Primer 1-1->6-1 B6(3-31) B7(3-31) D2(3-31) D4(3-31) D5(3-31) D7(3-31) D8(3-31) B3(4-01) B4(4-01) B6(4-01) C2(4-01) C5(4-01) C7(4-01) C8(4-01) POS(100pg) NTC
- Prepare diluted primers in PCR tubes
1rxn (8+1)x2 ->20rxns Primer mix (f+r 10 uM) 1 20 uL H2O 7 140 uL --------------------------------------
- Transfer 8 uL diluted primer into each reaction based on the arrangements
- Transfer 2 uL diluted templates into each reaction based on arrangements
- Prepare master mix in 1.5-mL microcentrifuge tube
1rxn (12+2)x8=112 rxns Taq (2X) 9.9 1108.8 SYBR (50X) 0.1 11.2 -------------------------------- 10.0 1120.0
- Mix well by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
- Gently vortex spin down the PCR reaction tubes. Place in Chromo4 machine to perform realtime PCR reaction.
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever