User:Floriane Briere/Notebook/CHEM-496/2011/09/20

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Objective

Today's objective is to mutate the GFP gene by using PCR technique.

Before being able to perform the PCR on a circular DNA plasmid we need to determine :

  • The primers' sequences (forward and reverse)
  • The temperature cycling program for the thermocycler
  • The volumes of solutions we are using during PCR

The circular DNA we're using is composed of 3600bp. The DNA molecule contains the GFP gene and other sequences which are mandatory to perform the PCR. The mutation we want to induce is to mute the GAT (which corresponds to an aspartic acid) onto a CAT (which corresponds to a cystein).

So, the DNA we want to create will have a cystein after the enterokinase cleavage site. The enterokinase cleavage site is used to cut the Nterm end of the protein so that we'll only get the protein we're interested in. To induce the mutation, we use primers which contain the mutation.

The vector we use is a PRSET/DNGFP vector; and the mutation we want to induce is called a D1CGFP mutation.

Protocol

To determine primers and the temperature cycling program we used:

* Determine the primers (forward and reverse):

Primers have to :

  • Have to be between 25 and 45 bases in length
  • Have a melting temperature (Tm) of ≥78°C (without the muted codon)
  • Have to mutation in their middle
  • Have a minimum GC content of 40% and should terminate in one or more C or G bases

We choose as the forward primer:

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

We choose as the reverse primer:

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

With Tm = 79.1°C (salt adjusted) and length = 36 nucleotids


To run the PCR, we are going to use already synthesized primers:

Forward primer (33 nucleotides; MJ Research Inc.): 5' GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA 3' Reverse primer (33 nucleotides; MJ Research Inc.): 5' TTC GGA TCC CCA CCA TCG ATC CTT ATC GTC ATC GTC 3'

GC content = 51.5% Melting Temperature = 64.6°C


* Determine the temperature cycling program for the thermocycler:

  1. 1 cycle: 30s at 95°C
  2. 16 cycles (because it's a single amino-acid change):
  • 30s at 95°C;
  • Then 1m at 55°C;
  • Then 3m36s at 68°C;
  1. 10m at 72°C;
  2. 24hours at 4°C.

* Determine which solutions we'll need to perform a PCR run:

  1. 5µl of reaction Buffer (10X)
  2. 1µl of template DNA (50ng/µl)
  3. 1.25µl of each primer (100ng/µl)
  4. 1µl of a dNTP mix (composed of 0.25µl of each dNTP whose concentration is 10mM)
  5. 40.5µl of double distilled water (to get a final volume of 50ml)
  6. 1µl of Pfu Turbo Enzyme (2.5U/µl; added at the last minute)
  7. 1µl of Wax solution (Bio Rad; to avoid the evaporation of the solution during the PCR)



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