Sam:LabNotes/Human/2009-2-27 exp2
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Perform MDA on human cell lysates - test 3 - Cell line GM18508[edit]
Samples & Materials[edit]
- Fresh lymphocytes, GM18508 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL
- UV-treated(10 min): 1X PBS, Ambion H2O, reagents in phi29 kit (10X Buffer, dTT, H2O)
Lymphocytes dilution[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 ---------------------------------------------------------- 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
Program setup[edit]
- Realtime PCR program set up (Total = 32 rxns)
- 0.5 cell lysate x 24
- Pos x 6 (gDNA-pos 1 ng/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1), 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
- H2O x 2
ALS and NS buffer preparation[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
- Aliquot ALS buffer and NS buffer to two individual PCR tube, with 100 uL of each buffer.
- UV treat NS and ALS buffer for 10 min.
MDA master mix (1.5-mL tube) preparation[edit]
1 rxn x 38 rxn ------------------------------------------------- H2O 6.2 uL 235.6 uL 200 uM N6 primer 5.0 uL 190.0 uL 10x RepliPhi phi-29 buffer 2.0 uL 76.0 uL 25mM dNTP 0.8 uL 30.4 uL 2X SYBR Green I 1.0 uL 38.0 uL RepliPhi Phi-29 (100U/ul) 1.0 uL 38.0 uL ------------------------------------------------- 608.0 uL (608/38=16) Leave on ice for later use
Prepare cell lysates[edit]
- Transfer 1 uL diluted cell (0.5 cell/uL) to each of 24 tubes in 8-well strips.
- Transfer Pos. and Neg. control on PCR tubes.
- 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.
Set up MDA reaction[edit]
- Add 16 uL MDA master 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:Sam022709-candidates.bmp Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction
- Pick-up 10 amplicons (Between 5 cell/uL and Blank): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7) for confirmation using regular PCR