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== Preparation == Thaw out all the reagents in Phi29 enzyme kit besides the actual enzyme. '''gDNA Template Dilution''' The following dilutions of gDNA were made 1. 1 ng/uL: 1uL gDNA/99uL H20 2. 30 pg/uL: 1.5 uL from (1)/ 48.5 uL H20 3. 3 pg/uL: 5 uL from (2)/ 45 uL H20 4. 300 fg/uL: 5 uL from (3)/ 45 uL H20 5. 30 fg/uL: 5 uL from (4)/ 45 uL H20 6. 3 fg/uL: 5 uL from (5)/ 45 uL H20 '''''Concentrations 2-6 were used, and 1 blank tube of water was used for a total of 6''''' '''N6 primer dilution''' Previously diluted by Sam '''KOH preparation''' 1. Weigh a KOH pellet and put into a 1.5 mL tube 2. Add nuclease free H20 to get a 5M conc. (Note: MW KOH=56.11) 3. Cap and vortex '''ALS Buffer Preparation''' 1. Add 82 uL H20, 10 uL 1M DTT, and 8 uL 5M KOH to 1.5 mL tube 2. Cap and vortex '''NS Buffer Preparation''' 1. Add 3 mL H20, 3 mL 2M Tris buffer, 4 mL 1N HCl to 15 mL tube 2. Vortex '''Master Mix Preparation''' Use the following reagents for the master mix. Prepare in a .5 mL tube. The master mix was made for a total of 14 reactions. 1. 86.8 uL H20 2. 28 uL 10X repliphi phi-29 buffer 3. 70 uL 200 uM N6 primer 4. 11.2 uL 25 nM dNTP 5. 14 uL 2X SYBR green 6. 14 uL Repliphi phi-29 Add the enzyme last To get volumes for a single reaction, divide by 14 '''Bio-Rad realtime PCR program''' Line 1: 30 C, 6 min Line 2: Plate read Line 3: Go to line 1 for additional 99 times Line 4: 85 C, 3 min Line 5: 4 C, forever Line 6: End
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