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

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==Objective==
==Objective==


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




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# Synthesize and purify two complimentary primers containing the desired mutation.
# Synthesize and purify two complimentary primers containing the desired mutation.
# Prepare reaction mixture with 5 μl of 10× reaction buffer, 1 μL of dsDNA template(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 41.75 μL of double-distilled water (ddH<sub>2</sub>O) to a final volume of 50 μL.
# Prepare reaction mixture with 5 μl of 10× reaction buffer, 1 μL of dsDNA template (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 41.75 μL of double-distilled water (ddH<sub>2</sub>O) to a final volume of 50 μL.
# Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
# Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
# Add 25 μL Bio Rad Chill Out™ liquid wax.
# Place reaction in thermocycler, for 1 cycle at 95°C for 30 s.
# Place reaction in thermocycler, for 1 cycle at 95°C for 30 s.
# Keep reaction mixture in thermocycler for 16 cycles as follows:
# Keep reaction mixture in thermocycler for 16 cycles as follows:
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##1 min at 55°C
##1 min at 55°C
##3.6 min at 68°C
##3.6 min at 68°C
#Place reaction mixture on ice for 2 min to cool to 37°C.
# Leave reaction mixture in thermocycler at 4°C for 24 hr.


==Data==
==Data==


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


*Forward Primer:
*Forward Primer:


::GAT AAG GAT GAC GAT AAG <b>TGT</b> CGA TGG GGA TCC GAA TTC GCC
::5'- GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC -3'


*Reverse Primer:
*Reverse Primer:


::GGC GAA TTC GGA TCC CCA TCG <b>ACA</b> CTT ATC GTC ATC CTT ATC  
::5'- GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC -3'
 
::(Mutation in bold)




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Primer actually used in experiment
Primer actually used in experiment:


*Forward Primer:
*Forward Primer:


::GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA
::5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3'


*Reverse Primer:
*Reverse Primer:


::TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC
::5'- TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC -3'


==Notes==
==Notes==

Revision as of 10:25, 1 November 2011

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Objective

To perform a PCR reaction to mutate green fluorescent protein (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, 1 μL of dsDNA template (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 41.75 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. Add 25 μL Bio Rad Chill Out™ liquid wax.
  5. Place reaction in thermocycler, for 1 cycle at 95°C for 30 s.
  6. 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
  7. Leave reaction mixture in thermocycler at 4°C for 24 hr.

Data

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

  • Forward Primer:
5'- GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC -3'
  • Reverse Primer:
5'- GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC -3'


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


Primer actually used in experiment:

  • Forward Primer:
5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3'
  • Reverse Primer:
5'- TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC -3'

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.




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