User:Laura Flynn/Notebook/Experimental Biological Chemistry/2011/09/20

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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.

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
    • 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:

Image:Cycle2011.jpg

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


Results

  • A suitable primer was researched for this experiment and determined to be:

Forward Primer:

GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC

Reverse Primer:

GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC

  • This primer was 39 base pairs, had a GC content of 51%, and had a salt adjusted melting temperature of 78.8°C.
  • The actual primer used was:

Forward Primer:

5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3'

Reverse Primer:

5'- TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC- 3'

  • This primer was 33 base pairs.


Notes


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