Sam:LabNotes/Microbiome-new/2009-6-30
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Human MDA test on 96-well plate-062909
Backgournd
- Test MDA amplification efficiency on 96-well plate with a scaled up reaction volumn.
Exp. Design
Preparation
- Thaw all the reagents needed for MDA
- 10X buffer (from kit)
- dNTP (from kit)
- Ambion RT-PCR H2O
- 1 M DTT (Sigma) – blue dot
- 0.5 M EDTA – blue dot
- Prepare fresh ALS buffer(400mM KOH, 100mM DTT, 10mM EDTA)
- Prepare 5M KOH
- Prepare ALS buffer (200 uL nuclease free H2O + 25 uL 1M DTT + 20 uL 5M KOH + 5 uL 0.5M EDTA)
- Test the pH value of ALS(10 uL) + NS buffer (10uL)mixture -> pH 7.5~8.0
- 10 min- UV treatment at crosslinker
- 20 uL RT-PCR grade H2O (Ambion) X1 - for Blank
- 20 uL ALS buffer X 3
- 20 uL NS buffer X 3
NOTE: After UV-treatment use a p200 pipettor to mix and pool the buffer together in 0.5-mL tubes.
- 10 min- UV treatment at working station
- Ethanol wipped pipettor and tools
- 96-well plate on the ice box.
- During the UV-treatment, set up Chromo4 (Bio-Rad)programming:
Line 1: 30 C, 6 min Line 2: Plate read Line 3: Goto line 2 for additional 99 times Line 4: 30 C, 6 min Line 5: Plate read Line 6: Goto line 4 for additional 99 times Line 7: 85 C, 3 min (to inactivate all of the enzymes left) Line 8: 15 C, forever Line 9: End
- Prepare 2X SYBR from the best 10,000X SYBR stock solution(1 uL of 200X SYBR + 99uL RT-PCR grade H2O)
- Prepare the master mix in a 1.5-mL tube wrapped with foil
1 rxn 8.5 rxns X 6 ------------------------------------------------------------- RT-PCR grade H2O 14.0 119.0 uL 10x RepliPhi phi-29 buffer 5.0 42.5 uL 1mM primer 2.5 21.25 uL 2X SYBR Green I 2.5 21.25 uL RepliPhi Phi-29 (100U/ul) 2.5 21.25 uL -------------------------------------------------------------- 26.5 225.25 uL (225.25/8.5=26.5) 25mM dNTP 2.0 17.0 uL - add dNTP later Leave the MDA master mix at RT
- Transfer 3.0 uL Template into wells.
- Transfer 3.0 uL ALS into wells. Mix by pipetting 4 times. Incubate 5 min on ice.
- Transfer 3.0 uL NS into wells.Mix by pipetting 4 times.
- Transfer 12.5 uL Tre into wells.Mix by pipetting 4 times.
!!!!!!!! Add dNTP into master mix!!!!!!!
- Transfer 28.5 uL complete master mix into each of reaction wells.
- Seal the 96-well plate with Bio-Rad qPCR film and place the reaction in Chromo4 qPCR machine. Program (using 20-hr incubation setting).
Results
File:96-well MDA.jpg
- The 50uL reaction volumn works well on 96-well plate. The master mix is insufficient for the last well (NTC control) and therefore there is a inconsistent reactions curve for the two NTC reactions. For 8 reaction master mix, prepare 9 reaction will be safer.
- The result also suggested that there is no obvious difference observed beteen fresh diluted 200pg and pre-diluted 200pg gDNA as the positive controls. These is no serious concern of DNA loss by sticking on the tube at the 200pg/uL level.