User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/20: Difference between revisions
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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> | |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> </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> </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. | |||
Procedure derived from [http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf|Stratagene QuikChange® Site-Directed Mutagenesis Kit] | |||
==Description== | |||
# 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== | ==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: | |||
::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). | |||
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 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectiveTo perform a PCR reaction to mutate green fluorescent protein (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site.
Description
DataPrimer determined using http://www.basic.northwestern.edu/biotools/oligocalc.html:
Primer actually used in experiment:
NotesPrimers 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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