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Sam:LabNotes/Microbione/2009-2-27/exp2
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='''Perform MDA on human cell lysates - test 3 - Cell line GM18508'''= ===Samples & Materials=== *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=== *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=== *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=== *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=== 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=== *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=== *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== [[Image:sam022709-candidates.bmp|600px]] '''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
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