Sam:LabNotes/Microbiome/2009-2-24: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>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) ...)
 
>Sam Chiang
(Removing all content from page)
 
Line 1: Line 1:
==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.

Latest revision as of 21:01, 16 March 2009