Sam:LabNotes/Human/2009-4-12
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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
Results
Primer 1-1 | |||||
Content | Description | C(t) | dCT(NTC-Sample) | Melting curve matched to Pos control | |
Sample | B6 (4/1) N6-No-Tre | 37.91 | -1.06 | n | |
Sample | B7 (4/1) N6-No-Tre | 7.72 | 29.13 | n | |
Sample | D2 (4/1) N6-No-Tre | 22.07 | 14.78 | n | |
Sample | D4 (4/1) N6-No-Tre | 6.37 | 30.48 | n | |
Sample | D5 (4/1) N6-No-Tre | 6.77 | 30.08 | n | |
Sample | D7 (4/1) N6-No-Tre | 7.37 | 29.48 | n | |
Sample | D8 (4/1) N6-No-Tre | 5.48 | 31.37 | n | |
Sample | B3 (3/31) L4N9-Tre | 36.3 | 0.55 | y | |
Sample | B4 (3/31) L4N9-Tre | 33.25 | 3.6 | y | |
Sample | B6 (3/31) L4N9-Tre | 34.64 | 2.21 | n | |
Sample | C2 (3/31) L4N9-Tre | 30.49 | 6.36 | y | |
Sample | C5 (3/31) L4N9-Tre | 4.76 | 32.09 | n | |
Sample | C7 (3/31) L4N9-Tre | 31.67 | 5.18 | y | |
Sample | C8 (3/31) L4N9-Tre | 32.3 | 4.55 | n | |
Sample | pos | 24.3 | |||
Blank | ntc | 36.85 | |||