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==Preparation== '''Thaw all of the reagents in Phi29 enzyme kit - except the pji29 enzyme" '''gDNA Template dilution''': Stock solution 100 ug/mL = 100 ng/uL Oligo tube (30 pg/uL = 10 human haplotype. 3 pg/uL = 1 human haplotype) Serial dilutioi: #Label 6 individual PCR tubes and fill with Nuclease-free H2O as following design #Perform the series dilution as followoing design ('''Note: Transferring by 20p pipettor; Mixing by 200p pipettor''') PCR tubes label '''1 ng/uL 30 pg/uL 3 pg/uL 300 fg/uL 30 fg/uL 3 fg/uL''' Trasnfer volume Stock sol. of gDNA -> 1.0 uL -> 1.5 uL -> 5.0 uL -> 5.0 uL -> 5.0 uL -> 5.0 uL H2O in PCR tubes 99.0 uL 48.5 uL 45.0 uL 45.0 uL 45.0 uL 45.0 uL ---------------------------------------------------------------------- Dilution factor 1/100 x 3/100 x 1/10 x 1/10 x 1/10 x 1/10 Conc. 100 ng/uL 1 ng/uL 30 pg/uL 3 pg/uL 300 fg/uL 30 fg/uL 3 fg/uL '''N6 primer dilution''': # Spin down the tube of N6 primer (261 nmole) # Add 131 uL H2O and mix -> totol conc. = 200 uM # Prepare a fresh 1.5 mL tube. # Perform a 1/200 dilution -> 1 uM N6-primer. Stay on ice for later use. '''Fresh 5M KOH (MW=56.11)''': # Weigh a KOH pellet and put into a fresh 1.5-mL tube. (pellet = 0.078 g = 1.39 x 10^(-3) mole) # Add nucleas-free H2O: 1.39 x 10^(-3) / 5 = 278 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''': # Prepare a 15 mL serological (scre cap) tube. # Add 3 mL H2O + 3 mL 2M Tris buffer + 4 mL 1N HCL in the tube. Mix with quick vortexing and spining down. (Total = 10 mL) '''phi29 reaction master mix: - for 16+2 rxns''' # Prepare a 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 18 rxn -------------------------------------------------------------- H2O 10.2 uL 183.6 uL 10X Repliphi phi-29 buffer 2.0 uL 36.0 uL 1uM N6 primer 1.0 uL 18.0 uL 25 nM dNTP 0.8 uL 14.4 uL 2X SYBR Green I 1.0 uL 18.0 uL Repliphi Phi-29 (100 U/uL) 1.0 uL 18.0 uL -------------------------------------------------------------- 16.0 uL 288.0 uL (288/18=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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