User:Elaine Marie Robbins/Notebook/CHEM-496/2011/11/01

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AuNP VII, Mutation of GFP II Main project page
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Objective

AuNP VII

To synthesize AuNP using the procedure developed here:


Bakshi, Mandeep S.; Kaur, Harpreet; Khullar, Poonam, Banipal, Tarlok, S.; Kaur, Gurinder; Singh, Narpinder Journal of Physical Chemistry C 2011, 115(7), 2982-2992

http://pubs.acs.org/doi/abs/10.1021/jp110296y


In addition, to repeat the experiment performed on 08/31/11, 09/07/11, 09/27/11, 09/28/11, 10/19/11, and 10/26/11.


Mutation of GFP II

To perform a PCR reaction to mutate green fluorescent protein (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site.

In addition, to repeat the experiment performed on 09/20/11.


Procedure derived from QuikChange® Site-Directed Mutagenesis Kit

Description

AuNP VII

  1. Combine 8 mL of water, 1 mL HAuCl4 (2.9 mM), and 1 mL BSA (15.5 μM) in that order a test tube.
  2. Combine 8 mL of water, 1 mL HCl (3 M), and 1 mL BSA in that order in a separate test tube.
  3. Place test tubes in an oven at 80°C for 30 min.
  4. Remove test tubes from oven for 10 min.
  5. Repeat steps 3 and 4 for 3.5 hr.


Mutation of GFP II

  1. Synthesize and purify two complimentary primers containing the desired mutation.
  2. 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.
  3. Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
  4. Add 25 μL Bio Rad Chill Out™ liquid wax.
  5. Keep reaction mixture in thermocycler for 18 cycles as follows:
    1. 30 s at 95°C
    2. 30 s at 60°C
    3. 7 min at 72°C
  6. Keep in thermocycler for 10 min at 72°C.
  7. Leave reaction mixture in thermocycler at 4°C for 24 hr.

Data

AuNP VII

At 60 minutes, green-gray fibers began to form. After 120 minutes, the fibers turned dark purple.


Mutation of GFP II

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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