User:Michael F. Nagle/Notebook/Chem 571/2012/10/24: Difference between revisions

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==Entry title==
==Objectives==
# 5μL of the DNA solution prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|last week]] was added to 40μL cells
*Continue expression of ADA
# The solution was placed on a heat block for 30 seconds and put on ice.
*Transform mutated plasmid to <i>E. Coli</i>
# The solution was incubated at 37<sup>o</sup>C and 225rpm for 1 hour
*Analyze 60-170 [Au/BSA] and [Au/Lysozyme] via UV/Vis
## 250μL Lysogeny Broth (LB) was added
==ADA expression==
# The solution was incubated again with the same specifications
* I came in several hours earlier than usual, at 9am, to resuspend cells in fresh LB.
 
* Cells were centrifuged at 4500rpm for 15 minutes.
# Solutions with Lysozyme and BSA prepared [[[[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|last week]]|last week]] were analyzed via UV/Vis. There was no peak at 520nm, so it was determined that heating at 50°C was insufficient. The solutions went back in the oven for 4 hours at 80°C, which is the same temperature BSA was seen succesfully unfolding at.
*Supernatant was dumped and 4mL fresh LB was added to one tube, which was shaken to resuspend cells before the solution was transferred to the next one, which was also shaken. This was repeated until all cells were resuspended.
*Cells were incubated at 160rpm and 37°C until 4pm.
*Cells were again centrifuged at 4500rpm for 15 minutes. Supernatant was dumped and cell swere resuspended in [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|binding buffer]] prepared by Puja Mody.
**Cells were stored at -80<sup>o</sup>C
==Transformation of Mutated Plasmid==
*[[AU_Biomaterials_Design_Lab:Protocols/Transformation_Protocol|Transformation protocol]] was followed, with the following modifications.
*1μL DPN1 was added to each plasmid solution.
*A vial of <i>E. Coli</i> was thawed on ice.
*10ng DNA in 5μL solution was added to 40μL cells.
*The cells were incubated in a 42<sup>o</sup>C water bath.
*After 30 seconds, they were removed and placed on ice.  
*200μL SOC media was added to one solution and 200μL LB was added to the other.
*Both were incubated at 275rpm at room temperature for an hour.
*25μL cells were added to one petri dish and 200μL to another.
*Both dishes contained LB and kanamycin.
*The plates were incubated overnight at 37<sup>o</sup>C
==UV/Vis of Au and BSA/Lysozyme==
# Solutions with Lysozyme and BSA prepared [[[[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|last week]] were analyzed via UV/Vis. There was no peak at 520nm, so it was determined that heating at 50°C was insufficient. The solutions went back in the oven for 4 hours at 80°C, which is the same temperature BSA was seen succesfully unfolding at.
# Solutions with Au and BSA prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|last week]] were analyzed via UV/Vis.
# Solutions with Au and BSA prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/16|last week]] were analyzed via UV/Vis.
==Data==
[[Image:Bsauvlast.png]]
[[Image:Lyzfirst.png]]
==Discussion==
*The results were similar to those of the [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/04|set analyzed 9/4]]. A drop to near-zero is seen at the mole ratio 130 [Au/BSA]
*The [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/11|set before this]] showed highest abs. at 130, and had a gradual drop until near-zero at 134.
*Since these data sets do not corroborate eachother, more trials are needed. So far only our second set corroborates data from Bakshi, et. al<sup>1</sup>


=References==
#Bakshi, M.S.; Kaur, H.; Khullar, P.; Banipal, T. S.l; Kaur, G.; Singh, N. Protein Films of Bovine Serum Albumen Conjugated Gold Nanoparticles: A Synthetic Route from Bioconjugated Nanoparticles to Biodegradable Protein Films.<i> J. Phys. Chem.</i> C, 2011, 115 (7), pp 2982–2992
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Revision as of 03:23, 7 December 2012

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Objectives

  • Continue expression of ADA
  • Transform mutated plasmid to E. Coli
  • Analyze 60-170 [Au/BSA] and [Au/Lysozyme] via UV/Vis

ADA expression

  • I came in several hours earlier than usual, at 9am, to resuspend cells in fresh LB.
  • Cells were centrifuged at 4500rpm for 15 minutes.
  • Supernatant was dumped and 4mL fresh LB was added to one tube, which was shaken to resuspend cells before the solution was transferred to the next one, which was also shaken. This was repeated until all cells were resuspended.
  • Cells were incubated at 160rpm and 37°C until 4pm.
  • Cells were again centrifuged at 4500rpm for 15 minutes. Supernatant was dumped and cell swere resuspended in binding buffer prepared by Puja Mody.
    • Cells were stored at -80oC

Transformation of Mutated Plasmid

  • Transformation protocol was followed, with the following modifications.
  • 1μL DPN1 was added to each plasmid solution.
  • A vial of E. Coli was thawed on ice.
  • 10ng DNA in 5μL solution was added to 40μL cells.
  • The cells were incubated in a 42oC water bath.
  • After 30 seconds, they were removed and placed on ice.
  • 200μL SOC media was added to one solution and 200μL LB was added to the other.
  • Both were incubated at 275rpm at room temperature for an hour.
  • 25μL cells were added to one petri dish and 200μL to another.
  • Both dishes contained LB and kanamycin.
  • The plates were incubated overnight at 37oC

UV/Vis of Au and BSA/Lysozyme

  1. Solutions with Lysozyme and BSA prepared [[last week were analyzed via UV/Vis. There was no peak at 520nm, so it was determined that heating at 50°C was insufficient. The solutions went back in the oven for 4 hours at 80°C, which is the same temperature BSA was seen succesfully unfolding at.
  2. Solutions with Au and BSA prepared last week were analyzed via UV/Vis.

Data

Discussion

  • The results were similar to those of the set analyzed 9/4. A drop to near-zero is seen at the mole ratio 130 [Au/BSA]
  • The set before this showed highest abs. at 130, and had a gradual drop until near-zero at 134.
  • Since these data sets do not corroborate eachother, more trials are needed. So far only our second set corroborates data from Bakshi, et. al1

References=

  1. Bakshi, M.S.; Kaur, H.; Khullar, P.; Banipal, T. S.l; Kaur, G.; Singh, N. Protein Films of Bovine Serum Albumen Conjugated Gold Nanoparticles: A Synthetic Route from Bioconjugated Nanoparticles to Biodegradable Protein Films. J. Phys. Chem. C, 2011, 115 (7), pp 2982–2992


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