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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> AuNP III, Protein Expression, DNA Transformation</span>
|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> AuNP III, Expression, DNA Transformation</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>}}
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==Objective==
==Objective==


To synthesize gold nanoparticles (Au NP) using the procedure developed here:  
<b>AuNP III</b>
 
To synthesize AuNP using the procedure developed here:  




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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]] and [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/07|09/07/11]].
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==
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<b>AuNP III</b>
<b>AuNP III</b>


# Combine 10 mL of 50 mM acetate buffer (pH 5.46) with 1.1×10<sup>-3</sup> g of .0015 mM bovine serum albumin (BSA) and 4.0×10<sup>-4</sup> g of 0.25 mM chloroauric acid (HAuCl<sub>4</sub>) in a capped test tube at room temperature.
# 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.
# Repeat with 10 mL of distilled water instead of acetate buffer.
# Take UV-vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
# Take UV vis spectra (200 nm to 800 nm) of buffer, water, and both reaction mixtures.
# Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
# Place reaction mixtures in an oven at 80°C for 6 hr under thermostatic conditions.
# Continue to take UV vis spectra of reaction mixture every 30 minutes.
# After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.
# After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.


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[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]
[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]]
# 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
# Prepare [[AU_Biomaterials_Design_Lab:Materials/LB|LB]] in a 250 mL Erlenmeyer flask.
## 0.875 g of LB
## 0.875 g of LB
## 35 mL of water
## 35 mL of water
# Cover the flask with foil
# Cover the flask with foil.
# [http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Autoclave| Autoclave]
# [http://openwetware.org/wiki/AU_Biomaterials_Design_Lab:Protocols/Autoclave| Autoclave].
# Allow the flask to cool
# Allow flask to cool.
# Add 35 μL of 1000X ampicillin
# Add 35 μL of 1000× ampicillin.
# Inoculate with the bacteria you are culturing using one of the two following methods
# Inoculate with the bacteria you are culturing by scraping frozen bacteria/glycerol mixture and adding to flask.
## Using sterile methods, retrieve a single colony from a petrie dish and drop it into the flask
# Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm.
## Using sterile methods, add 5uL of a glycerol stock to the flask
# Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm




<b>DNA Transformation</b>
<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.




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
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Objective

AuNP III

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 08/31/11 and 09/07/11.


Protein Expression

To begin culturing bacteria to express inteins.


DNA Transformation

To transform DNA coding for mutant GFP (mutated on 09/20/11) into Novablue cells.

Description

AuNP III

  1. 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 HAuCl4 in a capped test tube at room temperature.
  2. Take UV-vis spectra (200 nm to 800 nm) of a sample of reaction mixture at 70°C every 30 min.
  3. Place reaction mixture in an oven at 80°C for 2.5 hr under thermostatic conditions.
  4. After 2.5 hr, remove from oven and cool to room temperature; leave sitting overnight.


Protein Expression

Starter Culture Media, Protein Expression

  1. Make expression culture media.
  2. Prepare LB in a 250 mL Erlenmeyer flask.
    1. 0.875 g of LB
    2. 35 mL of water
  3. Cover the flask with foil.
  4. Autoclave.
  5. Allow flask to cool.
  6. Add 35 μL of 1000× ampicillin.
  7. Inoculate with the bacteria you are culturing by scraping frozen bacteria/glycerol mixture and adding to flask.
  8. Place the flask in a shaker/incubator and culture overnight at 37°C and 200 rpm.


DNA Transformation

  1. Digest non-methylated DNA with 1 μL Dpnl.
  2. Make LB/agar plates with ampicillin.
  3. Place sterile tube and DNA in ice bucket for 15 min.
  4. Add 5 μL of DNA and 40 μL of cells to the bottom of the tube.
  5. Incubate on ice for 30 min.
  6. Heat shock DNA/cells at 42°C for 30 s.
  7. Incubate on ice for 5 min.
  8. Add 250 μL of SOC media.
  9. Incubate in shaker at 37°C for 1 hr.
  10. Spread 100 μL of cells on LB/agar plate.
  11. Store plate (inverted) in 37°C oven overnight.

Data

AuNP 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.


DNA Transformation

Plate did not appear to be successful; no cells grew.

Notes

The AuNP solution turned from clear to purple while in the oven, indicating that AuNPs were actually formed.




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