Sam:LabNotes/Microbiome-new/2009-6-30

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Revision as of 07:18, 30 June 2009 by >Sam Chiang (New page: ='''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 ...)
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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.