User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/09/19: Difference between revisions
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#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. | #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. | ||
# 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. | ||
# Incubate the expression cultures at | # Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6. | ||
# Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG|IPTG]] to each flask in order to induce protein expression | # Add 1mL of 0.4M [[AU_Biomaterials_Design_Lab:Materials/IPTG|IPTG]] to each flask in order to induce protein expression | ||
# Continue shaking for 3-4 hours | # Continue shaking for 3-4 hours | ||
# Add 30 mL of the binding buffer into each flask | |||
# Harvest the cells by centrifuging at 4500rpm for 15 minutes | # Harvest the cells by centrifuging at 4500rpm for 15 minutes | ||
# collect the cells and place in -80°C freezer | |||
==Calculations== | |||
Theoretical calculations | * Binding Buffer: 1 liter total solution at pH 7.4. | ||
a. Theoretical calculations | |||
#20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris. | #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. | ** similar calculations were done for all compounds added into the buffer solution. | ||
# .5M NaCl= 29.2195 g NaCl | # .5M NaCl= 29.2195 g NaCl | ||
# 30mM Imidazole | # 30mM Imidazole= 2.0424 g Imidazole | ||
* Elution Buffer: 500 mL total solution at pH 7.4 | |||
a. Theoretical calculations | |||
#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. | |||
# .5M NaCl= 14.609 g NaCl | |||
#.5 M imidazole= 17.019 g imidazole. | |||
==Conclusions== | ==Conclusions== |
Revision as of 01:43, 26 September 2012
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Protein Expression.Goals
Procedure
Calculations
a. Theoretical calculations
a. Theoretical calculations
Conclusions
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