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==Preparation== '''Thaw all of the reagents in Phi29 enzyme kit - except the phi29 enzyme''' '''Human Jurkat cell gDNA dilution''' # Prepare the gDNA dilution as described in 02-06-09' experiment. # Only 30 pg/uL, 300 fg/uL and 3 fg/uL were used in this exp. '''2X SYBR Green I dilution''': # Dlute the SYBR solution from 10,000X to 2X. Wrap the 2X SYBR with foil and store it in 4 degree. '''N6 primer dilution''': # Use the diluted 200 uM N6 primer from 02-06-09' experiment. '''Fresh 5M KOH (MW=56.11)''': # Weigh a KOH pellet and put into a fresh 1.5-mL tube. pellet = 0.065 g = 1.158 x 10^(-3) mole # Add nucleas-free H2O: 1.158 x 10^(-3) / 5 = 232 uL # Cap the tube and mix by slight vortexing. Label and date the tube '''ALS buffer''': - use for dissoicating the dsDNA # Prepare a fresh 1.5-mL tube. # Add 82 uL H2O + 10 uL 1 M DTT + 8 uL 5M KOH in the tube. Mix with quick vortexing and spining down. (Total = 100 uL) (p.s the reagent DTT (1 M) is supplied in the phi29 enzyme kit) '''NS buffer''': # Use the one from 02-06-09' experiments. '''phi29 reaction master mix: - for 4+1 rxns''' # Prepare two fresh 0.5 mL tube. # Prepare the master mix as following recipt. # Mix with quick vortexing and spining down (Total = 288 uL ). Leave on ice for later use. 1 rxn 5 rxn -------------------------------------------------------------- 200 uM N6 primer 5.0 uL 25.0 uL 10X Repliphi phi-29 buffer 2.0 uL 10.0 uL H2O 6.2 uL 31.0 uL 25 nM dNTP 0.8 uL 4.0 uL 2X SYBR Green I 1.0 uL 5.0 uL --(New and old 2X SYBR for master mix 1 and 2, respectively) Repliphi Phi-29 (100 U/uL) 1.0 uL 5.0 uL -------------------------------------------------------------- 16.0 uL 80.0 uL (80/5=16) '''Bio-Rad realtime PCR programing''' Set up the program as following Line 1: 30 C, 6 min Line 2: Plate read Line 3: Goto line 1 for additional 99 times Line 4: 85 C, 3 min (to kill all of the enzymes left) Line 5: 4 C, forever Line 6: End
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