User:Madeleine Y. Bee/Notebook/CHEM-572 2014S/2014/04/01

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Current revision (13:05, 1 April 2014) (view source)
 
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Cell/Scaffold Tests</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Scaffold Prep, and AgNPs without BSA</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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*From [http://pubs.acs.org/doi/pdf/10.1021/ed084p322 Synthesis and Study of Silver Nanoparticles]
*From [http://pubs.acs.org/doi/pdf/10.1021/ed084p322 Synthesis and Study of Silver Nanoparticles]
*10 ml of 1 mM AgNO3 was added dropwise (about 1 drop/sec) to 30 ml of 2 mM NaBH4 (cooled in ice bath), while the reaction mixture was vigorously stirred using a magnetic stir plate.  Entire procedure takes about 3 minutes.  
*10 ml of 1 mM AgNO3 was added dropwise (about 1 drop/sec) to 30 ml of 2 mM NaBH4 (cooled in ice bath), while the reaction mixture was vigorously stirred using a magnetic stir plate.  Entire procedure takes about 3 minutes.  
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=====With BSA (direct from article)=====
 
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*From [http://www.sciencedirect.com/science/article/pii/S0021979712009526 Characterization of Silver/Bovine Serum Albumin (Ag/BSA) nanoparticles structure: Morphological, compositional, and interaction studies]
 
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*"Initially 0.9 g of BSA was mixed with 40 ml of deionized water. One milli liter of 0.423 M silver nitrate was added to BSA solution at ∼23 °C and sonicated for 10 min. This was followed by the addition of 1 ml of 0.423 M of sodium borohydride and sonicated for additional 30 min to reduce the silver ions in the solution. The resulting brown homogeneous solution was centrifuged at 21,500 rpm and the residue was washed with ethanol, suspended in water, and saved in an amber colored bottle at 4 °C."
 
===Sample Preparation: Bacteria Culture===
===Sample Preparation: Bacteria Culture===
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*Autoclave
*Autoclave
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*Cell tests
 
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===Type I Collagen Test (Detection and Quantification)===
 
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*[http://www.chondrex.com/products/mouse-type-i-collagen-detection-kit Company procedure ]
 
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===Collagen Staining Procedure & Microscope Imaging===
 
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*[http://www.chondrex.com/products/sirius-red-fast-green-collagen-staining-kit Company procedure]
 
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===Trypsin Cell Detachment Procedure & Cell Counting===
 
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*[http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/celldetachmentprotocol.pdf Company procedure]
 
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Current revision

Scaffold Prep, and AgNPs without BSA Main project page
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April 1, 2014

Objectives

  • Making new scaffolds
  • New AgNP procedures

Au:Myoglobin Nanofibers: Sample Prep for Cell Culture

Image:2014_0211_myo_bsa_NPs.PNG

  • Solutions:
    • TRIS dopamine: 0.0475g dopamine hydrochloride and 0.03g TRIS in 25mL to make 10mM solution at pH 8.46
    • Au: 0.011g in 10mL to make 2.793mM solution
    • Myoglobin: 0.011g in 10mL to make 0.0621mM solution

Plain Scaffolds

  • 4 PLA scaffolds printed for each test tube
  • Three test tubes filled with scaffolds and water
  • Autoclaved

Brute Force Nanofiber-coated Scaffolds

  • 4 PLA scaffolds printed for each test tube
  • Gold:Myoglobin (100:1) nanofibers synthesized by above method
  • Nanofibers dried onto PLA scaffolds
  • Scaffolds submerged in water
  • Autoclaved

Original Method for Nanofiber-coated Scaffolds

  • 4 PLA scaffolds printed for each test tube
  • Gold:Myoglobin (100:1) nanofibers synthesized by above procedure with scaffolds in solution
  • Autoclaved

Polydopamine-coated Scaffolds

  • 4 PLA scaffolds printed for each test tube
  • Scaffolds submerged in 10mM pH 8.46 TRIS dopamine hydrochloride solution for 1 hour
  • Add 1mL of 0.0621 Myoglobin to each test tube, shake tube to combine, allow surface functionalization to occur for 5 hours
  • Rinse with deionized water and allow scaffold to dry overnight
  • Scaffolds submerged in water
  • Autoclaved

Polydopamine Nanofiber-coated Scaffolds

  • 4 PLA scaffolds printed for each test tube
  • Scaffolds submerged in 10mM pH 8.5 TRIS dopamine hydrochloride solution for 1 hour
  • Add 1mL of 0.0621 Myoglobin to each test tube, shake tube to combine, allow surface functionalization to occur for 5 hours
  • Rinse with deionized water and allow scaffold to dry overnight
  • Gold:Myoglobin (100:1) nanofibers synthesized by above procedure with polydopamine-coated scaffolds in solution
  • Autoclaved

Breakdown

Today
  • Time sensitive: Make 40mL TRIS-polydopamine solution
  • Time sensitive: Make 6 tests tubes of TRIS-polydopamine solution, put scaffolds in all 6 test tubes at 12 p.m. (3 for pD only, 3 for pD-original)
  • Make Au and Myoglobin solutions
  • Make 9 test tubes of 100:1 Au:Myo Nanofibers (3 for original, 3 for brute force fibers, 3 for pD-original tomorrow; see volumes in chart above)
  • Time sensitive: At 1 p.m. add 1mL of 0.0621M Myoglobin to all 6 tubes (3 for pD only, 3 for pD-original)
  • Put scaffolds into 3 of the 9 Au:Myo test tubes when finished (3 for original)
  • Make 3 test tubes of water -> put scaffolds into 3 test tubes when finished
  • Put 9 test tubes (3 brute force, 3 original, 3 water) into oven, set for 4 hours at 80C
  • Time Sensitive: At 6 p.m. rinse pD scaffolds that have soaked for 5 hours, submerge 6 in water overnight (if possible, submerge 3 of 6 tubes in Au:Myo solution and put all 6 in oven for 4 hours), dump Au:Myo fibers onto scaffolds for brute force
Tomorrow
  • If oven was not finished yesterday: submerge 3 pD scaffolds into original solution, leave other 3 in water and put in oven for 4 hours at 80C
  • Submerged dried brute force scaffolds in water
  • Autoclave all: 3 plain, 3 brute force, 3 original, 3 pD-Myo, 3 pD-Myo-Au

Sample Preparation: AgNPs

Without BSA
  • From Synthesis and Study of Silver Nanoparticles
  • 10 ml of 1 mM AgNO3 was added dropwise (about 1 drop/sec) to 30 ml of 2 mM NaBH4 (cooled in ice bath), while the reaction mixture was vigorously stirred using a magnetic stir plate. Entire procedure takes about 3 minutes.

Sample Preparation: Bacteria Culture

  • Make solution
  • Autoclave



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