Sam:LabNotes/Human/2009-2-27 exp2

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Perform MDA on human cell lysates - test 3 - Cell line GM18508[edit]

Samples & Materials[edit]

  • Fresh lymphocytes, GM18508 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL
  • UV-treated(10 min): 1X PBS, Ambion H2O, reagents in phi29 kit (10X Buffer, dTT, H2O)


Lymphocytes dilution[edit]

  • Take cultured lymphocytes suspension from one T25 flask
  • Aliquot 100 uL suspension for cell counting using the cell counter (Beckman)
  • Withdraw the desired cell amount (2 x 10^5 cells) and wash using UV-treated 1XPBS twice (Spin at 1000rpm for 3min)
  • Resuspend in 1 mL UV-treated PBS
  • Make dilution to 10 cell/uL using UV-treated PBS:
   Dilution Factor     Start      1/10        1/40      1/400     
   ----------------------------------------------------------
   BAC mixture         100 uL     100 uL     10 uL      10 uL
   PBS                            900 uL     30 uL      90 uL
   ----------------------------------------------------------
   Concentration       200/uL      20/uL      5/uL     0.5/uL
  
   Mix by repeat pipetting using p200 pipettor in each step
  

Program setup[edit]

  • Realtime PCR program set up (Total = 32 rxns)
    • 0.5 cell lysate x 24
    • Pos x 6 (gDNA-pos 1 ng/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1), 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
    • H2O x 2


ALS and NS buffer preparation[edit]

  • 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 8 ul 5M KOH, 2 ul 0.5M EDTA, 10 ul 1M DTT, 80 ul nuclease free H2O
    • 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
    • Aliquot ALS buffer and NS buffer to two individual PCR tube, with 100 uL of each buffer.
    • UV treat NS and ALS buffer for 10 min.


MDA master mix (1.5-mL tube) preparation[edit]

                                1 rxn      x 38 rxn
  -------------------------------------------------
  H2O                            6.2 uL   235.6 uL
  200 uM N6 primer               5.0 uL   190.0 uL
  10x RepliPhi phi-29 buffer     2.0 uL    76.0 uL
  25mM dNTP                      0.8 uL    30.4 uL 
  2X SYBR Green I                1.0 uL    38.0 uL
  RepliPhi Phi-29 (100U/ul)      1.0 uL    38.0 uL
  -------------------------------------------------
                                          608.0 uL   (608/38=16)
  Leave on ice for later use


Prepare cell lysates[edit]

  • Transfer 1 uL diluted cell (0.5 cell/uL) to each of 24 tubes in 8-well strips.
  • Transfer Pos. and Neg. control on PCR tubes.
  • 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.


Set up MDA reaction[edit]

  • Add 16 uL MDA master 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.


Results[edit]

  File:Sam022709-candidates.bmp
  Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction
  • Pick-up 10 amplicons (Between 5 cell/uL and Blank): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7) for confirmation using regular PCR