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

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


*1% Agarose Gel was prepared by mixing 25 mL of tris acetate ethylene tetracetic acid buffer with 0.25 grams of agarose. This solution was microwaved for about 40 seconds and the poured into the electrophoresis chamber and set to cool for 20 minutes. After it was properly cooled, the comb and wedges were removed and the gel was covered in the buffer that was used previously.
*1μl of the sample was combined with 5μl of gel loading dye on paraffin wax, and then was pipetted up and down to ensure proper distribution. A gel ladder consisting of DNA ladder, glycerol, and blue dye was loaded in well 1, and then each 6μl sample of protein and loading dye was distributed to each well. Well 3 contained the sample prepared for this experiment.
*After all samples were loaded, the gel electrophoresis machine was turned on and set to 80 volts. It ran for about 40 minutes before it was turned off and the gel was removed from the machine. The gel was then stained with a mixture of TAE and dilute ethidium bromide for 10 minutes. It was rinsed with a mixture of TAE and then observed under UV light.


==Results==
==Results==
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*This primer was 33 base pairs.
*This primer was 33 base pairs.


[[Image:DNAgel.jpg]]
DNA was shown present in well 3, which is where the DNA from this experiment was placed. The mark is shown around the 3000 bp mark, indicating that the correct cutting occurred in the DNA.
Analysis of the DNA sequence was conducted at an off-site sequencing business, and it was shown that there was no mutation in the DNA, as it was identical to the initial DNA.


==Notes==
==Notes==

Revision as of 12:16, 30 November 2011

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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:

  • 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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