Sam:LabNotes/Human/2009-4-12: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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'''Primer 2-1''' | '''Primer 2-1''' | ||
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| Blank||ntc||32.83|||| | | Blank||ntc||32.83|||| | ||
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'''Primer 3-1''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Ct value''' | |||
| align="center" style="background:#f0f0f0;"|'''dCT(NTC-Sample)''' | |||
| align="center" style="background:#f0f0f0;"|'''Melting curve matched to Pos control''' | |||
|- | |||
| Sample||B6 (4/1) N6-No-Tre||41.68||-3.51||y | |||
|- | |||
| Sample||B7 (4/1) N6-No-Tre||N/A||N/A||n | |||
|- | |||
| Sample||D2 (4/1) N6-No-Tre||30.77||7.4||n | |||
|- | |||
| Sample||D4 (4/1) N6-No-Tre||28.89||9.28||n | |||
|- | |||
| Sample||D5 (4/1) N6-No-Tre||26.03||12.14||n | |||
|- | |||
| Sample||D7 (4/1) N6-No-Tre||33.34||4.83||y | |||
|- | |||
| Sample||D8 (4/1) N6-No-Tre||33.09||5.08||y | |||
|- | |||
| Sample||B3 (3/31) L4N9-Tre||36.87||1.3||n | |||
|- | |||
| Sample||B4 (3/31) L4N9-Tre||33.8||4.37||y | |||
|- | |||
| Sample||B6 (3/31) L4N9-Tre||N/A||N/A||y | |||
|- | |||
| Sample||C2 (3/31) L4N9-Tre||34.13||4.04||n | |||
|- | |||
| Sample||C5 (3/31) L4N9-Tre||40.4||-2.23||y | |||
|- | |||
| Sample||C7 (3/31) L4N9-Tre||28.02||10.15||n | |||
|- | |||
| Sample||C8 (3/31) L4N9-Tre||39.56||-1.39||y | |||
|- | |||
| Sample||pos||23.83|||| | |||
|- | |||
| Blank||ntc||38.17 | |||
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Revision as of 20:39, 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
Results
Primer 1-1
Content | Description | Ct value | 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 | 3.60 | 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 | ||
Primer 2-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 37.18 | -4.35 | n |
Sample | B7 (4/1) N6-No-Tre | 30.78 | 2.05 | n |
Sample | D2 (4/1) N6-No-Tre | 22.67 | 10.16 | n |
Sample | D4 (4/1) N6-No-Tre | 29.83 | 3 | y |
Sample | D5 (4/1) N6-No-Tre | 33 | -0.17 | n |
Sample | D7 (4/1) N6-No-Tre | 26.69 | 6.14 | y |
Sample | D8 (4/1) N6-No-Tre | 34.23 | -1.4 | y |
Sample | B3 (3/31) L4N9-Tre | 31.56 | 1.27 | y |
Sample | B4 (3/31) L4N9-Tre | 24.07 | 8.76 | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C2 (3/31) L4N9-Tre | 26.25 | 6.58 | y |
Sample | C5 (3/31) L4N9-Tre | N/A | N/A | n |
Sample | C7 (3/31) L4N9-Tre | 30.57 | 2.26 | y |
Sample | C8 (3/31) L4N9-Tre | 37.38 | -4.55 | n |
Sample | pos | 23.26 | ||
Blank | ntc | 32.83 | ||
Primer 3-1
Content | Description | Ct value | dCT(NTC-Sample) | Melting curve matched to Pos control |
Sample | B6 (4/1) N6-No-Tre | 41.68 | -3.51 | y |
Sample | B7 (4/1) N6-No-Tre | N/A | N/A | n |
Sample | D2 (4/1) N6-No-Tre | 30.77 | 7.4 | n |
Sample | D4 (4/1) N6-No-Tre | 28.89 | 9.28 | n |
Sample | D5 (4/1) N6-No-Tre | 26.03 | 12.14 | n |
Sample | D7 (4/1) N6-No-Tre | 33.34 | 4.83 | y |
Sample | D8 (4/1) N6-No-Tre | 33.09 | 5.08 | y |
Sample | B3 (3/31) L4N9-Tre | 36.87 | 1.3 | n |
Sample | B4 (3/31) L4N9-Tre | 33.8 | 4.37 | y |
Sample | B6 (3/31) L4N9-Tre | N/A | N/A | y |
Sample | C2 (3/31) L4N9-Tre | 34.13 | 4.04 | n |
Sample | C5 (3/31) L4N9-Tre | 40.4 | -2.23 | y |
Sample | C7 (3/31) L4N9-Tre | 28.02 | 10.15 | n |
Sample | C8 (3/31) L4N9-Tre | 39.56 | -1.39 | y |
Sample | pos | 23.83 | ||
Blank | ntc | 38.17 | ||