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

From OpenWetWare
Jump to navigationJump to search
Mutation and Amplification of GFP <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>



Objective

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


Procedure derived from QuikChange® Site-Directed Mutagenesis Kit

Description

  1. Synthesize and purify two complimentary primers containing the desired mutation.
  2. Prepare reaction mixture with 5 μl of 10× reaction buffer, 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:
    1. 30 s at 95°C
    2. 1 min at 55°C
    3. 3.6 min at 68°C
  6. Place reaction mixture on ice for 2 min to cool to 37°C.

Data

Primer determined using http://www.basic.northwestern.edu/biotools/oligocalc.html

  • 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

Primers must have a melting point Tm ≥ 78°C, a GC content ≥ 40%, and be between 25-45 bases in length.

Sequence of GFP vector provided by Invitrogen.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/20&oldid=539170"