User:Elaine Marie Robbins/Notebook/CHEM-496/2011/11/01: Difference between revisions
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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> AuNP VII, Mutation | |style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> AuNP VII, Mutation of GFP II</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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In addition, to repeat the experiment performed on [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/08/31|08/31/11]], [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/07|09/07/11]], [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/27|09/27/11]], [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/28|09/28/11]], [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/19|10/19/11]], and [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/26|10/26/11]]. | In addition, to repeat the experiment performed on [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/08/31|08/31/11]], [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/07|09/07/11]], [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/27|09/27/11]], [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/28|09/28/11]], [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/19|10/19/11]], and [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/10/26|10/26/11]]. | ||
<b>Mutation of GFP II</b> | |||
To perform a PCR reaction to mutate green fluorescent protein (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site. | |||
In addition, to repeat the experiment performed on [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/20|09/20/11]]. | |||
Procedure derived from [http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf|Stratagene QuikChange® Site-Directed Mutagenesis Kit] | |||
==Description== | ==Description== | ||
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<b>AuNP VII</b> | <b>AuNP VII</b> | ||
# Combine | # Combine 8 mL of water, 1 mL HAuCl<sub>4</sub> (2.9 mM), and 1 mL BSA (15.5 μM) in that order a test tube. | ||
# Combine 1 mL | # Combine 8 mL of water, 1 mL HCl (3 M), and 1 mL BSA in that order in a separate test tube. | ||
# Place test tubes in an oven at 80°C for 30 min. | # Place test tubes in an oven at 80°C for 30 min. | ||
# Remove test tubes from oven for 10 min. | # Remove test tubes from oven for 10 min. | ||
# Repeat steps 3 and 4 for 3.5 hr. | # Repeat steps 3 and 4 for 3.5 hr. | ||
<b>Mutation of GFP II</b> | |||
# 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 μL of oligonucleotide control primer #1 (100 ng/μL), 1 μl of oligonucleotide control primer #2 (100 ng/μL), 1 μl of dNTP mix (10mM), and 41 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. | |||
# Keep reaction mixture in thermocycler for 18 cycles as follows: | |||
##30 s at 95°C | |||
##30 s at 60°C | |||
##7 min at 72°C | |||
# Keep in thermocycler for 10 min at 72°C. | |||
# Leave reaction mixture in thermocycler at 4°C for 24 hr. | |||
==Data== | ==Data== | ||
<b>AuNP VII</b> | |||
At 60 minutes, green-gray fibers began to form. After 120 minutes, the fibers turned dark purple. | |||
<b>Mutation of GFP II</b> | |||
Primers used in this experiment: | |||
*Forward primer: | |||
::5'- TAC GAC GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC -3' | |||
*Reverse Primer: | |||
::5'- GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC GTC GTA- 3' | |||
==Notes== | ==Notes== |
Revision as of 11:29, 15 November 2011
AuNP VII, Mutation of GFP II | <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> |
ObjectiveAuNP VII To synthesize AuNP using the procedure developed here:
http://pubs.acs.org/doi/abs/10.1021/jp110296y
To perform a PCR reaction to mutate green fluorescent protein (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site. In addition, to repeat the experiment performed on 09/20/11.
DescriptionAuNP VII
DataAuNP VII At 60 minutes, green-gray fibers began to form. After 120 minutes, the fibers turned dark purple.
Primers used in this experiment:
Notes
|