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

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span>
|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Mutation and Amplification of GFP</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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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]
Perform PCR to mutate GFP to contain a cysteine residue just after the enterokinase cleavage site.


==Description==
==Description==
Materials
[https://docs.google.com/document/pub?id=1q9liaSkDZ_35N6F_ztcXqKOu6g1w6qxqpCQ-tD8zOVM GFP plasmid sequence from Invitrogen]


[http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf Stratagene Quick Change PCR manual]
# 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.
# 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.
# 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
# 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, T<sub>m</sub>=78.8°C (salt adjusted).
 
 


What you need to do today:
Primer actually used in experiment:
#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 [http://www.basic.northwestern.edu/biotools/oligocalc.html 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 [[AU_Biomaterials_Design_Lab:Protocols|protocols page]]


==Data==
*Forward Primer:
* Add data and results here...


==Notes==
::5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3'
[http://www.youtube.com/watch?v=x5yPkxCLads PCR song]


*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

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 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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