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

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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.




==Objective==
Procedure derived from [http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf|Stratagene QuikChange® Site-Directed Mutagenesis Kit]
 
To perform a PCR reaction to mutate Green Fluorescent Proten (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site.


==Description==
==Description==


# 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 (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 (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==


Forward Primer:
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:


::GAT AAG GAT GAC GAT AAG <b>TGT</b> CGA TGG GGA TCC GAA TTC GCC
::5'- GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC -3'


Reverse Primer:


::GGC GAA TTC GGA TCC CCA TCG <b>ACA</b> CTT ATC GTC ATC CTT ATC
::39bp, 51% GC content, T<sub>m</sub>=78.8°C (salt adjusted).


(Mutation in bold)




39bp, 51% GC content, T<sub>m</sub>=78.8°C (salt adjusted).
Primer actually used in experiment:


==Notes==
*Forward Primer:


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


*Reverse Primer:


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


[[Category:Course]]
==Notes==
[[Category:Miscellaneous]]


Primers must have a melting point T<sub>m</sub> ≥ 78°C, a GC content ≥ 40%, and be between 25-45 bases in length.


Sequence of GFP vector provided by [https://docs.google.com/document/pub?id=1q9liaSkDZ_35N6F_ztcXqKOu6g1w6qxqpCQ-tD8zOVM| Invitrogen].


[[Category:Course]]
[[Category:Course]]

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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