Sam:LabNotes/Microbiome/2009-2-24
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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.