User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/09/20

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Contents

Objective

  • Perform Polymerase Chain Reaction (PCR) to mutate Green Fluorescent Protein (GFP) to include a cysteine residue after the enterokinase cleavage site
  • Method's for experiment were based off "QuickChange Site-Directed Mutagenesis Kit": [1]

Description

  • Primers were determined using the following link: [2]
  • Reaction mixture was prepared using the following:

42 μL dH2O

5 μL 10x reaction buffer

1 μL dsDNA template (50 ng/μL)

1.25 μL Primer 1

1.25 μL Primer 2

1 μL dNTP mix (10 mM)

  • 1 μL of PfuTurbo DNA Polymerase (2.5 Units/μL) was then added to reaction mixture
  • 25 μL of Bio Rad liquid wax was added to the top of the mixture
  • Reaction mixture was placed into thermocycler with the following cycles:

1 cycle 95 Degrees Celsius for 30 seconds

15 cycles as follows:

- 30 sec at 95 Degrees Celsius

- 1 min at 55 Degrees Celsius

-3.6 min at 68 Degrees Celsius

Final cycle 4 Degrees Celsius for 24 hours

Data

  • Theoretical Primers

Forward Primer

5' - CGA CGA TGA CGA TAA GCG ATG GGG ATC CGA ATT CGC - 3'

Reverse Primer

5' - GCG AAT TCG GAT CCC CAT CGC TTA TCG TCA TCG TCG - 3'

56% G/C Content

Melting Temperature - Tm = 79.1 Degrees Celsius (Salt Adjusted)


  • Actual Primers

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'

Melting Temperature - Tm > 78 Degrees Celsius

G/C % > 40%

Notes

  • The GFP plasmid was 3600 base pairs in length
  • The mutation involved transforming the GAT codon located after the enterokinase clevage in the GFP plasmid.
  • The vector used in this experiment is the PRSET/DNGFP vector. The mutation is a D1CGFP mutation.
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