User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/11/01

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Objective

The purpose of this experiment is to use PCR to mutate GFP so that a cysteine residue can replace the aspartic acid residue directly after the enterokinase cleavage site on the vector. AuNP synthesis was also preformed. This time, the order that the components were being added was analyzed. It was: water, HAuCl4, and then BSA.

Procedure

  • Two complimentary primers (listed below) containing the desired mutation were synthesized and purified.
  • A sample reaction was prepared as follows:
    • 5 μl of 10× reaction buffer)
    • 1.25 μl of dsDNA template (Wildtype GFP)
    • 1 μl of the forward primer
    • 1 μl of the reverse primer
    • 1 μl of dNTP mix
    • double-distilled water (ddH2O) to a final volume of 50 μl
  • At the end just before temperature cycling, 1 μl of PfuTurbo DNA polymerase and 25μl of wax were added to the sample.
  • Each reaction was placed underwent specific cycling parameters, which were as follows:

  • The reaction was also cycled for 10 minutes at 72°C and for 24 hours at 4°C.

AuNP:

Protocol was followed from the previous week. Two test tubes were used:

  • Test tube one: 1 mL of BSA, 1 mL of HAuCl4, and 8 mL of water
  • Test tube two: 1mL of BSA and 1 mL of 3 M HCl, and 8 mL of water

Both were kept in an oven at approximately 70 degrees for thirty minute cycles, with 10 minute breaks at room temperature in between.


Results

AuNP:

In test tube one, there was aggregation that formed dark green matter at the top of the test tube.

In test tube two, there was no aggregation or visible change.

Neither of the results were expected. This could be due to contamination of the HAuCl4 stock.

Primers:

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'

  • This primer was 39 base pairs.

Notes

  • T=0 occured at 12:45 pm.
  • At T=30 minutes an aggregation of AU+ ions was observed at the top of the test tube one.
  • The concentration of BSA was 1.55 microM. The concentration of HAuCl4 was 2.89 mM.



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