Perform PCR to mutate GFP to contain a cysteine residue just after the enterokinase cleavage site.
GFP plasmid sequence from Invitrogen
Stratagene Quick Change PCR manual
What you need to do today:
- Get a better understanding for PCR
- Design forward and reverse primers for the plasmid in order to meet the requirements laid out in the Quick Change Manual
- A "calculator" to determine the melting temperature of oligonucleotides can be found here. When performing the calculation leave out the nucleotide bases that you are mutating. (These bases are going to be mismatched during the PCR runs).
- Figure out what solutions you will need to perform a PCR run and what volumes of each solution you will need. You will need to check on the stock solutions that we have to make these calculations.
- Design a temperature cycling program for the thermocycler.
- Run a PCR reaction with the primers and DNA plasmid that I already have.
- Write a generic protocol for PCR on the our protocols page
- Add data and results here...
inner life of a cell
Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.