Sam:LabNotes/Human/2009-4-12: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang |
||
Line 17: | Line 17: | ||
Column 1 -> 6 Column 7 -> 12 | Column 1 -> 6 Column 7 -> 12 | ||
Primer 1-1->6-1 Primer 1-1->6-1 | Primer 1-1->6-1 Primer 1-1->6-1 | ||
B6( | B6(4-01) B7(4-01) | ||
D2( | D2(4-01) D4(4-01) | ||
D5( | D5(4-01) D7(4-01) | ||
D8( | D8(4-01) B3(3-31) | ||
B4( | B4(3-31) B6(3-31) | ||
C2( | C2(3-31) C5(3-31) | ||
C7( | C7(3-31) C8(3-31) | ||
POS(100pg) NTC | POS(100pg) NTC | ||
Revision as of 01:27, 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, pos (gDNA 100pg/uL), NTC(H2O)
- 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(4-01) B7(4-01) D2(4-01) D4(4-01) D5(4-01) D7(4-01) D8(4-01) B3(3-31) B4(3-31) B6(3-31) C2(3-31) C5(3-31) C7(3-31) C8(3-31) POS(100pg) NTC
- Prepare diluted primers in PCR tubes
1rxn (8+1)x2 ->20rxns Primer mix (f+r 10 uM) 1.0 20.0 uL H2O 7.0 140.0 uL ---------------------------------------- 8.0 160.0
- 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 the master mix by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
- Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately 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