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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''' {| {{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||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''' {| {{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||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''' {| {{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 |- | |} '''Primer 4-1''' {| {{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||N/A||N/A||n |- | Sample||B7 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||D2 (4/1) N6-No-Tre||N/A||N/A||y |- | Sample||D4 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||D5 (4/1) N6-No-Tre||N/A||N/A||y |- | Sample||D7 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||D8 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||B3 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||B4 (3/31) L4N9-Tre||38.75||N/A||y |- | Sample||B6 (3/31) L4N9-Tre||N/A||N/A||n |- | Sample||C2 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||C5 (3/31) L4N9-Tre||N/A||N/A||n |- | Sample||C7 (3/31) L4N9-Tre||N/A||N/A||n |- | Sample||C8 (3/31) L4N9-Tre||N/A||N/A||n |- | Sample||pos||23.51||N/A|| |- | Blank||ntc||N/A||N/A |- | |} '''Primer 5-1''' {| {{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||40.66||-8.72||n |- | Sample||B7 (4/1) N6-No-Tre||44.13||-12.19||n |- | Sample||D2 (4/1) N6-No-Tre||37.36||-5.42||n |- | Sample||D4 (4/1) N6-No-Tre||36.8||-4.86||n |- | Sample||D5 (4/1) N6-No-Tre||32.06||-0.12||y |- | Sample||D7 (4/1) N6-No-Tre||38.86||-6.92||n |- | Sample||D8 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||B3 (3/31) L4N9-Tre||40.66||-8.72||n |- | Sample||B4 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||B6 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||C2 (3/31) L4N9-Tre||36.89||-4.95||n |- | Sample||C5 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||C7 (3/31) L4N9-Tre||40.25||-8.31||n |- | Sample||C8 (3/31) L4N9-Tre||N/A||N/A||n |- | Sample||pos||24.02|||| |- | Blank||ntc||31.94 |- | |} '''Primer 6-1''' {| {{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||38.26||0.89||n |- | Sample||B7 (4/1) N6-No-Tre||34.57||4.58||y |- | Sample||D2 (4/1) N6-No-Tre||42.1||-2.95||n |- | Sample||D4 (4/1) N6-No-Tre||39.49||-0.34||n |- | Sample||D5 (4/1) N6-No-Tre||33.99||5.16||y |- | Sample||D7 (4/1) N6-No-Tre||N/A||N/A||n |- | Sample||D8 (4/1) N6-No-Tre||28.96||10.19||y |- | Sample||B3 (3/31) L4N9-Tre||34.64||4.51||y |- | Sample||B4 (3/31) L4N9-Tre||39.92||-0.77||y |- | Sample||B6 (3/31) L4N9-Tre||35.97||3.18||y |- | Sample||C2 (3/31) L4N9-Tre||36.08||3.07||y |- | Sample||C5 (3/31) L4N9-Tre||N/A||N/A||y |- | Sample||C7 (3/31) L4N9-Tre||33.26||5.89||y |- | Sample||C8 (3/31) L4N9-Tre||37.54||1.61||n |- | Sample||pos||26.85|||| |- | Blank||ntc||39.15 |- | |}
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