User:Nader A. Khamis/Notebook/Experimental Biological Chemistry AU Fall 2011/2011/09/20

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Objective

  • To able to select primers of proper length and GC content.
  • Use PCR to mutate Green Flourescence Protein in order to insert a cysteine residue after the enterokinase cleavage site.
  • Experimental procedures were based off "QuickChange Site-Directed Mutagenesis Kit": [1]

Description

  • The following link was used to determine the primers: [2]


  • 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'

Melting Temperature of Theoratical Primers (Tm) = 79.1°C Percent of GC content= 56%.

  • The actual experiment involved the following

Forward primer used was:5' GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA 3'

Reverse primer used was: 5' TTC GGA TCC CCA CCA TCG ATC CTT ATC GTC ATC GTC 3'

Melting Temperature of actual primers = 64.6°C Percent of GC content = 51.5%

  1. The following procedure was executed to prepare solutions needed for PCR.

5 uL 10x reaction buffer

42 uL dH2O

1 uL dsDNA template (50 ng/uL)

1.25 uL Primer 1

1.25 uL Primer 2

1 uL dNTP mix (10 mM)

  1. Approximately 6.1µl of Pfu Turbo DNA Polymerase (2.5Units/µl)was added to reaction mixture.
  2. Approximately 25ul of Bio Rad liquid wax was pipetted on the top of the reaction mixture.
  3. The reaction mixture was placed into the thermal cycler and the following setup was made.
  • 1 cycle at 95°C
  • 15 cycles containing the following:
  1. 30 sec at 95 Degrees Celsius
  2. 1 min at 55 Degrees Celsius
  3. 3.6 min at 68 Degrees Celsius

The final cycle was: 4°C for 24 hours.

Data

  • DNA sequence was obtained on October 12th, 2011.

Notes

  • Size of GFP Plasmid: 3600bp.

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