Sam:LabNotes/Microbiome-new/2009-6-30: Difference between revisions

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>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 ...)
 
>Sam Chiang
(Removing all content from page)
 
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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==
[[Image:96-well MDA.jpg|400px]]
*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.

Latest revision as of 07:19, 30 June 2009