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)

  1. 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.
  2. Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
  3. Add 25 μL Bio Rad Chill Out™ liquid wax.
  4. 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


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