User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19

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# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
# Inoculate the [[AU_Biomaterials_Design_Lab:Protocols/Expression_Culture_Media|expression culture media]] by dividing the resuspended cells among the Fernbach flasks.
--[[User:Abigail E. Miller|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning.  
--[[User:Abigail E. Miller|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning.  
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# Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.
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That afternoon:
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# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG|IPTG]] to each flask in order to induce protein expression
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# 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG|IPTG]] was added to each flask in order to induce protein expression. The 4 fernbach flasks were then allowed to continue to shake for 3 more hours.
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# Continue shaking for 3-4 hours
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#The cells were harvested by centrifuging at 4500rpm for 15 minutes. 2 runs of this had to be done because not all the media was able to fit in the centrifuge jars in one run.  
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# Add 30 mL of the binding buffer into each flask
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# The cells were then resuspended using 30mL of binding buffer for flasks 2-4 and 10mL separate for flask 1.
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# Harvest the cells by centrifuging at 4500rpm for 15 minutes
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# These flasks were then placed in a -80°C freezer overnight.
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# collect the cells and place in -80°C freezer
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--[[User:Abigail E. Miller|Abigail E. Miller]] 10:53, 7 October 2012 (EDT)this is too much like the protocol. what did you actually do?  at what time was the IPTG added? you did not measure the OD at 600nm. what time did you harvest the cells? therefore how long were they allowed to express?  how many times did the cells have to be spun down? what deviations did you make from the protocol - I know you separated flask 1 form the other three. that is not clear here.
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==Calculations==
==Calculations==

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Protein Expression.

Goals

  • Induce Protein expression
  • make the binding buffer and the elution buffer
  • Harvest the cells that were grown.

Procedure

  • Protein Expression
  1. This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB. For more details on the process in the morning, see Mary's Notebook
  2. Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.

--Abigail E. Miller 10:53, 7 October 2012 (EDT)it is not clear that this was done in the morning. That afternoon:

  1. 1mL of 0.4M IPTG was added to each flask in order to induce protein expression. The 4 fernbach flasks were then allowed to continue to shake for 3 more hours.
  2. The cells were harvested by centrifuging at 4500rpm for 15 minutes. 2 runs of this had to be done because not all the media was able to fit in the centrifuge jars in one run.
  3. The cells were then resuspended using 30mL of binding buffer for flasks 2-4 and 10mL separate for flask 1.
  4. These flasks were then placed in a -80°C freezer overnight.

Calculations

  • Binding Buffer: 1 liter total solution at pH 7.4.

a. Theoretical calculations

  1. 20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris.
    • similar calculations were done for all compounds added into the buffer solution.
  1. .5M NaCl= 29.2195 g NaCl
  2. 30mM Imidazole= 2.0424 g Imidazole
  • Elution Buffer: 500 mL total solution at pH 7.4

a. Theoretical calculations

  1. 20mM Tris= .020 × .500= .01 mols × 121.14/ 1mol= 1.2114 g Tris
    • similar calculations/stoichiometry was done for all compounded added into the buffer solution.
  1. .5M NaCl= 14.609 g NaCl
  2. .5 M imidazole= 17.019 g imidazole.
  • Refer to Mary's Notebook for the actual amounts of buffer components measured out and added to solution.

--Abigail E. Miller 10:54, 7 October 2012 (EDT)did you have to adjust the pH?

Conclusions

  • sample 1 looked a little different when the samples were looked at this morning prior to centrifuging. It was a different shade of yellow in the erlenmeyer flask. As a result, for the rest of the protein expression, it will be separate from the other 3 made samples just in case there was anything amiss in that sample (sample 1).



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