Sam:LabNotes/Human/2009-2-25 exp2

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Perform MDA on human cell lysates - test 2[edit]

  • 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[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     
   ----------------------------------------------------------
   Cell suspension     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
  
   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: 0.5-cell x 24
    • Positive controls: 6 No-template control: 2
    • Total reaction: 32 x 20ul

Set up MDA experiment[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


  • 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 36 rxn
  -------------------------------------------------
  H2O                            6.2 uL   223.2 uL
  200 uM N6 primer               5.0 uL   180.0 uL
  10x RepliPhi phi-29 buffer     2.0 uL    72.0 uL
  25mM dNTP                      0.8 uL    28.8 uL 
  2X SYBR Green I                1.0 uL    36.0 uL
  RepliPhi Phi-29 (100U/ul)      1.0 uL    36.0 uL
  -------------------------------------------------
                                          576.0 uL   (576/36=16)
  Leave on ice for later use
  
  • Prepare cell lysates:
    • Transfer 1 uL diluted cell (0.5 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: 8 reactions
    • Transfer 1 uL of 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
    • gDNA 1 ug/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1)
    • DNAse-free H2O (x 2)


  • 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.


Results[edit]

  File:Sam022509-graph only2-with annotation.bmp
  Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction

Discussion[edit]

  • Nine out of 24 reactions (with 0.5 cell/uL template) were pick up for regular PCR confirmation
    • A3, A6, A7, B4, B7, C3, C4 and C8


  • One Blank reaction (D8) failed due to insufficient Master mix. Need to increase the master mix volume for this design.


  • The back ground amplification (Ct=26~28)still occur in Blank control, althoough the UV-treated H2O (from phi29 kit) was used as template.
    • ALS and NS buffer were UV-treated. All of the phi29 reagents kit except dNTP and enzyme were UV-treated (10 min).
    • It is possible the contamination is from the Master mix.