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
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==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   
    ----------------------------------------------------------
    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
 
    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==
  [[Image:sam022509-graph only2-with annotation.bmp|600px]]
  '''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.

Latest revision as of 21:04, 16 March 2009