Sam:LabNotes/Microbiome/2009-2-25/exp2: Difference between revisions

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>Sam Chiang
(New page: ==Perform MDA on human cell lysates - test 2== *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...)
 
>Sam Chiang
Line 15: Line 15:
     Dilution Factor    Start      1/10        1/40      1/400     
     Dilution Factor    Start      1/10        1/40      1/400     
     ----------------------------------------------------------
     ----------------------------------------------------------
     BAC mixture        100 uL    100 uL    10 uL      10 uL
     Cell suspension    100 uL    100 uL    10 uL      10 uL
     PBS                            900 uL    30 uL      90 uL
     PBS                            900 uL    30 uL      90 uL
     ----------------------------------------------------------
     ----------------------------------------------------------
Line 30: Line 30:
**Positive controls: 6  No-template control: 2
**Positive controls: 6  No-template control: 2
**Total reaction: 32 x 20ul
**Total reaction: 32 x 20ul


===Set up MDA experiment===
===Set up MDA experiment===

Revision as of 17:09, 11 March 2009

Perform MDA on human cell lysates - test 2

  • 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 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

  • 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

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

Discussion

  • 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.