AlanFung:LabNotes/Human MDA Halotyping/2009-7-20
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PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)
Procedures
- Experiment design
- Primers: Human genome specific primers set 1 (primer 1 to 12)
- Templates: 200pg MDA amplicons from 06/16/09 200pg MDA: L4N7, N6 in 1/50 dilution. Pos (100 pg/uL gDNA), NTC (RT-PCR grade H2O)
- Prepare diluted primers in PCR tubes (24 tubes)
For each primer needs 7+1 reaction 1rxn (7+1) rxns Primer mix (f+r 10 uM) 1.0 8.0 uL H2O 7.0 56.0 uL --------------------------------------------- 8.0 64.0 uL
- Prepare master mix in 1.5-mL microcentrifuge tube, wrapped with foil
1rxn (12+2) rxns x 7 Econo (2X) 10.0 140.0 Template 2.0 28.0 – (N6, 15C, 20C, 1-100dNTP, 1-1000dNTP, Pos, H2O) ----------------------------------- 12.0 168.0 168/14=12
- Mix well the master mix by vortexing. Transfer 12 uL Master mix into each of 12 reactions.
- Transfer 8 uL diluted primer into each reaction based on the arrangements
- 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 60 sec) x 50 -> 72C 10min -> 15C hold
PCR test on the whole set of human genome primers
Background
- Testing if the human genome primers (f+r 10uM premixed) were degraded.
Procedures
- Primers: Human genome specific primers set 1 (primer 1 to 24)
- Templates: Pos (1 ng/uL gDNA)
1rxn Primer mix (f+r 10 uM) 1.0 uL H2O 7.0 uL Template 2.0 uL Econo (2X) 10.0 uL --------------------------------------------- 20.0 uL Amplified at 52C for 40 cycles
Gel electrophoresis
- 110mL (0.5X TBE) 2% agarose + 8 uL SYBR safe -> 2 middle gel with 26-well comb
- Loading: Sample 7uL + 6X loading buffer 1.5 uL (Order: P1 -> P24 left to right)
- Run at 135V for 20 min
File:ZhangLab 2 2009-07-16 14hr 23min pos-test - annotate.jpg
- All of the human genome primers are still working fine. 40 cycles amplification with 7uL sample loading amount will be used as the standard for following PCR tests