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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 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.
To determine primers and the temperature cycling program we used:
* Determine the primers (forward and reverse):
Primers have to :
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
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 which solutions we'll need to perform a PCR run: