User:Michaela Harper/Notebook/Biological Chemistry Lab/2011/11/01

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Objective To repeat PCR procedure from 09/20/11 and to repeat gold nanoparticle synthesis in order to determine under what conditions purple fibers are formed. Description PCR 10× Reaction Buffer: 100 mM KCl 100 mM(NH4)2SO4 200 mM Tris-HCl (pH 8.8) 20 mM MgSO4 1% Triton X-100 1 mg/ml nuclease-free bovine serum albumin (BSA) SAMPLE: 5 μl of 10× reaction buffer X μl (5–50 ng) of dsDNA template X μl (125 ng) of oligonucleotide primer #1 X μl (125 ng) of oligonucleotide primer #2 1 μl of dNTP mix ddH2O to a final volume of 50 μl Then add: 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) Prepare reaction mixture with 5 μl of 10× reaction buffer, 1 μL of dsDNA template(50 ng/μL), 1 μL of oligonucleotide control primer #1 (100 ng/μL), 1 μl of oligonucleotide control primer #2 (100 ng/μL), 1 μl of dNTP mix (10mM), and 41 μL of double-distilled water (ddH2O) to a final volume of 50 μL. Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture. Add 25 μL Bio Rad Chill Out™ liquid wax. Design a temperature cycling program for the thermocycler. Temperature cycling: Segment 1: 1 cycle, 95 degrees, 30 seconds Segment 2: 16 cycles, 95 degrees, 30 seconds 55 degrees, 1 minute 68 degrees, 3.6 minutes (for 1min/kb of plasmid, 3600 bps of plasmid) Leave reaction mixture in thermocycler at 4°C for 24 hr. Nanoparticles Add 8 mL of water to a test tube, then 1 mL of gold solution (2.89 mM), then 1 mL of BSA solution (1.55 μM). This will test if the order the solutions are added has an effect on the formation of the purple fibers. Repeat with 3 M HCl instead of gold solution. Keep in oven at approximately 70°C for 30-minute increments, removing the solutions for 10 minutes in between increments. Data Nanoparticles At 60 minutes, green-gray fibers began to form. After 120 minutes, the fibers turned dark purple. PCR Primers used in this experiment: Forward primer: 5'- TAC GAC GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC -3' Reverse Primer: 5'- GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC GTC GTA- 3' Notes