User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/27: Difference between revisions
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|style="background-color: # | |style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> AuNP III, Expression, DNA Transformation</span> | ||
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==Objective== | |||
<b>AuNP III</b> | |||
To synthesize AuNP using the procedure developed here: | |||
Bakshi, Mandeep S.; Kaur, Harpreet; Khullar, Poonam, Banipal, Tarlok, S.; Kaur, Gurinder; Singh, Narpinder Journal of Physical Chemistry C 2011, 115(7), 2982-2992 | |||
http://pubs.acs.org/doi/abs/10.1021/jp110296y | |||
In addition, to repeat the experiment performed on [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/08/31|08/31/11]] and [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/07|09/07/11]]. | |||
<b>Protein Expression</b> | |||
To begin culturing bacteria to express inteins. | |||
<b>DNA Transformation</b> | |||
To transform DNA coding for mutant GFP (mutated on [[User:Elaine_Marie_Robbins/Notebook/CHEM-496/2011/09/20| 09/20/11]]) into Novablue cells. | |||
==Description== | ==Description== | ||
<b>AuNP III</b> | |||
# Combine 10 mL of 50 mM acetate buffer (pH 5.46) with 0.974 mL of 0.0015 mM BSA and 0.581 mL of 0.25 mM HAuCl<sub>4</sub> in a capped test tube at room temperature. | |||
# Take UV-vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min. | |||
# Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions. | |||
# After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight. | |||
<b>Protein Expression</b> | |||
[http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Starter_Culture_Media| Starter Culture Media], [http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Protein_Expression| Protein Expression] | |||
# Make [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media|expression culture media]]. | |||
# Prepare [[AU_Biomaterials_Design_Lab:Materials/LB|LB]] in a 250 mL Erlenmeyer flask. | |||
## 0.875 g of LB | |||
## 35 mL of water | |||
# Cover the flask with foil. | |||
# [http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Autoclave| Autoclave]. | |||
# Allow flask to cool. | |||
# Add 35 μL of 1000× ampicillin. | |||
# Inoculate with the bacteria you are culturing by scraping frozen bacteria/glycerol mixture and adding to flask. | |||
# Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm. | |||
<b>DNA Transformation</b> | |||
# Digest non-methylated DNA with 1 μL Dpnl. | |||
# Make LB/agar plates with ampicillin. | |||
# Place sterile tube and DNA in ice bucket for 15 min. | |||
# Add 5 μL of DNA and 40 μL of cells to the bottom of the tube. | |||
# Incubate on ice for 30 min. | |||
# Heat shock DNA/cells at 42°C for 30 s. | |||
# Incubate on ice for 5 min. | |||
# Add 250 μL of SOC media. | |||
# Incubate in shaker at 37°C for 1 hr. | |||
# Spread 100 μL of cells on LB/agar plate. | |||
# Store plate (inverted) in 37°C oven overnight. | |||
==Data== | ==Data== | ||
<b>AuNP III</b> | |||
The absorbance values for the AuNP solution were subtracted from the absorbance values for a blank acetate buffer to create a set of corrected absorbance values. The corrected absorbance values at 550 nm were graphed against time. The graph shows a positive linear relationship, indicating that AuNPs were formed in solution. | |||
<center>[[Image:Absorbance vs time 9-27-11.jpg|600px]]</center> | |||
In addition, a second graph showing corrected absorbance against wavelength was created, to show the absorbance levels across all of the wavelengths measured by the UV-vis. | |||
<center>[[Image:Absorbance vs wavelength 9-27-11.jpg|600px]]</center> | |||
A magnified version of this graph shows that absorbance values at around 550 nm increased over time, indicating the creation of AuNPs. | |||
<center>[[Image:Absorbance vs wavelength magnified 9-27-11.jpg|600px]]</center> | |||
<b>DNA Transformation</b> | |||
Plate did not appear to be successful; no cells grew. | |||
==Notes== | ==Notes== | ||
The AuNP solution turned from clear to purple while in the oven, indicating that AuNPs were actually formed. | |||
[[Category:Course]] | [[Category:Course]] | ||
[[Category:Miscellaneous]] | [[Category:Miscellaneous]] | ||
Revision as of 09:44, 26 October 2011
AuNP III, Expression, DNA Transformation | <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 III To synthesize AuNP using the procedure developed here:
http://pubs.acs.org/doi/abs/10.1021/jp110296y
To begin culturing bacteria to express inteins.
To transform DNA coding for mutant GFP (mutated on 09/20/11) into Novablue cells. DescriptionAuNP III
Starter Culture Media, Protein Expression
DataAuNP III The absorbance values for the AuNP solution were subtracted from the absorbance values for a blank acetate buffer to create a set of corrected absorbance values. The corrected absorbance values at 550 nm were graphed against time. The graph shows a positive linear relationship, indicating that AuNPs were formed in solution. In addition, a second graph showing corrected absorbance against wavelength was created, to show the absorbance levels across all of the wavelengths measured by the UV-vis. A magnified version of this graph shows that absorbance values at around 550 nm increased over time, indicating the creation of AuNPs.
Plate did not appear to be successful; no cells grew. NotesThe AuNP solution turned from clear to purple while in the oven, indicating that AuNPs were actually formed.
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