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
- Combine 8 mL of water, 1 mL HAuCl4 (2.9 mM), and 1 mL BSA (15.5 μM) in that order a test tube.
- Combine 8 mL of water, 1 mL HCl (3 M), and 1 mL BSA in that order in a separate test tube.
- Place test tubes in an oven at 80°C for 30 min.
- Remove test tubes from oven for 10 min.
- Repeat steps 3 and 4 for 3.5 hr.
Mutation of GFP II
- Synthesize and purify two complimentary primers containing the desired mutation.
- 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.
- Keep reaction mixture in thermocycler for 18 cycles as follows:
- 30 s at 95°C
- 30 s at 60°C
- 7 min at 72°C
- Keep in thermocycler for 10 min at 72°C.
- 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:
- 5'- TAC GAC GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC -3'
- 5'- GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC GTC GTA- 3'
Notes
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