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==Procedures== *Thaw the reagents on ice and UV-treated H2O, 1XPBS and equipment for 10 min *The the cool block out from frezor and leave in room tempterature for at least 10 min (To prevent the over-cold problem) *Dilute the gDNA in UV-treated H2O into 3ng/uL, 300pg/uL, 30pg/uL, 3pg/uL *Prepare ALS buffer and test the pH (use old NS buffer is ok) *Transfer 250 uL(or enough amount) of ALS, NS, and dH2O into PCR tubes and UV-treated for 10 min (using cross linking machine) *At the same time, dilute the cell with UV-treated PBS into 10 cell/uL, 5 cell/uL and 0.5 cell/uL *Prepare the 2X SYBR (2uL of 50X SYBR + 48 uL H2O) *Prepare MDA master mix (in a 0.6-mL tube)and leave on ice 1 rxn x 36 rxn ------------------------------------------------- H2O 6.2 uL 223.2 uL 1mM L4N6 primer 1.0 uL 36.0 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 ------------------------------------------------- 432.0 uL (432/36=12) *Prepare cell lysates: **Trasfer 1 uL ALS buffer into each of 32 PCR tubes on the cool-block **Transfer 1 uL template (Pos, Neg, diluted whole cell) into each of 32 PCR tubes as prevous indication, mix with pipetting in each transferring. **Incubate for 5 min on cool block **Transfer 1 uL NS buffer into each of 32 PCR tubes, mix with pipetting. *Transfer 5 uL of Tre(1.2M) into each of 32 PCR tubes, mix with pipetting. *Transfer 12 uL MDA master mix to each of 32 PCR tubes. Mix with pipetting. *Perfomr realtime PCR in Chromo4 with the new program line 1. Incubate at 30C for 6 min line 2. Plate read line 3. Goto line 1 for 99 more times line 4. Incubate at 30C for 6 min line 5. Plate read line 6. Goto line 4 for 99 more times line 7. Incubate at 85C for 3 min line 8. Incubate at 4C forever Note: Tre treatment will delay the reaction time, so we put 200 cycles in total. (The maximum setting is 99 cycles for the program) *Stop the program (when the reaction reach the signal saturation) and pick up the samples *Inactivate the samples at 85C for 3 min using thermocycler.
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