Sam:LabNotes/Microbiome/2009-2-24

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Revision as of 19:06, 8 March 2009 by >Sam Chiang (New page: ==Perform MDA on human cell lysates== *Samples: **Fresh lymphocytes, GM18507 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL **UV-treated (10 min) 1X PBS - 10 mL (prepared in 15-mL Corning tube) ...)
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Perform MDA on human cell lysates

  • Samples:
    • Fresh lymphocytes, GM18507 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL
    • UV-treated (10 min) 1X PBS - 10 mL (prepared in 15-mL Corning tube)


Lymphocytes pre-processing

  • Take 1/4 of cultured lymphocytes from one T25 flask;
    • Spin at 1000rpm for 3min;
    • Remove culture medium;
    • Resuspend the cell pellet in 5ml 1x PBS;
    • Perform cell counting;
    • Make dilution to 10 cell/uL using UV treated PBS:
   Dilution Factor     Start      1/10      1/100     1/1000  x 4
   -----------------------------------------------------------
   Lymphocytes         100 uL     100 uL     10 uL      10 uL 
   PBS                            900 uL     90 uL      90 uL
   -----------------------------------------------------------
   Concentration       200/uL      20/uL     2/uL      0.2 uL
  
   Mix by repeat pipetting using p200 pipettor in each step
  
   To decrease the error in the last dilution (1/100 -> 1/1000), this step is performed in four replicates, and labeled #1~#4


  • Realtime PCR program set up (Number of reactions)
    • cell lysate: 0.2-cell x 32
    • Positive controls: 2 No-template control: 2
    • Total reaction: 36 x 20ul

Set up MDA experiment

  • Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
    • Mix Add 82 uL H2O + 10 uL 1 M DTT + 8 uL 5M KOH in the tube.
    • Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8


  • Decontamination with UV-treatment
    • Aliquot ALS buffer and NS buffer to two individual PCR tube, with 100 uL of each buffer.
    • Treat the buffer with UV for 10 min.


  • Prepare MDA master mix (in a 1.5-mL tube):
                                1 rxn      x 40 rxn
  -------------------------------------------------
  H2O                            6.2 uL     248 uL
  200 uM N6 primer               5.0 uL     200 uL
  10x RepliPhi phi-29 buffer     2.0 uL      80 uL
  25mM dNTP                      0.8 uL      32 uL 
  2X SYBR Green I                1.0 uL      40 uL
  RepliPhi Phi-29 (100U/ul)      1.0 uL      40 uL
  -------------------------------------------------
                                            640 uL   (640/40=16)
  Leave on ice for later use
  
  • Prepare cell lysates:
    • Transfer 1 uL diluted cell (0.2 cell/uL) to each of 32 tubes in four 8-well strips. Diluted cell #1 goes to strip #1, and so on for the rest (#2 -> #2 strip...).


  • Prepare Pos. and Neg. control:
    • Transfer 1 uL of 20 cell/uL sample (x 1) , 2 cell/uL sample (x 1), DNAse-free H2O (x 2) into four PCR tubes.


  • DNA denaturing and nutralization:
    • Add 1.5 uL ALS buffer into 1 uL diluted cells and mix by repeat pipetting (~3 times), incubate at RT for 3 min or until all tubes are finished (Time it!)
    • Add 1.5 uL NS buffer, mix by repeat pipetting. Place the tubes on cool rack.


  • Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.


  • Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.