User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/20: Difference between revisions

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Forward Primer:
Forward Primer:


GAT AAG GAT GAC GAT AAG <b>TGT</b> CGA TGG GGA TCC GAA TTC GCC
::GAT AAG GAT GAC GAT AAG <b>TGT</b> CGA TGG GGA TCC GAA TTC GCC


Reverse Primer:
Reverse Primer:


GGC GAA TTC GGA TCC CCA TCG <b>ACA</b> CTT ATC GTC ATC CTT ATC  
::GGC GAA TTC GGA TCC CCA TCG <b>ACA</b> CTT ATC GTC ATC CTT ATC  


Mutation in bold.
(Mutation in bold)


39bp, 51% GC content, T<sub>m</sub>=78.8°C (salt adjusted),
39bp, 51% GC content, T<sub>m</sub>=78.8°C (salt adjusted),

Revision as of 11:04, 20 September 2011

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Objective

To perform a PCR reaction to mutate Green Fluorescent Proten (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site.

Description

  1. Synthesize and purify two complimentary primers containing the desired mutation.
  2. Prepare reaction mixture with 5 μl of 10× reaction buffer (2.5 U/μL), 2 μL of pWhitescript 4.5-kb control plasmid (50 ng/μL), 1.25 μL of oligonucleotide control primer #1 (100 ng/μL), 1.25 μl of oligonucleotide control primer #2 (100 ng/μL), 1 μl of dNTP mix (10mM), and 39.5 μL of double-distilled water (ddH2O) to a final volume of 50 μL.
  3. Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
  4. Place reaction in thermocycler, for 1 cycle at 95°C for 30 s.
  5. Keep reaction mixture in thermocycler for 16 cycles as follows:
  • 30 s at 95°C
  • 1 min at 55°C
  • 3.6 min at 68°C
  1. Place reaction mixture on ice for 2 min to cool to 37°C.

Data

Forward Primer:

GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC

Reverse Primer:

GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC

(Mutation in bold)

39bp, 51% GC content, Tm=78.8°C (salt adjusted),

Notes

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.




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