User:Matt Hartings/Notebook/Photosynthesis/2012/06/19: Difference between revisions
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==Objective== | ==Objective== | ||
Continue sperm whale myoglobin expression as started by Tamra [[User:Tamra_L._Fisher/Notebook/Research_for_Dr._Hartings/2012/06/18|notebook page]] and finish purification of protein as started yesterday [[ | Continue sperm whale myoglobin expression as started by Tamra [[User:Tamra_L._Fisher/Notebook/Research_for_Dr._Hartings/2012/06/18|notebook page]] and finish purification of protein as started yesterday [[User:Matt_Hartings/Notebook/Photosynthesis/2012/06/18|notebook page]]. | ||
==Description== | ==Description== | ||
# Add | |||
==Start WT Hemoglobin Expression== | |||
#Inoculate starter cultures (35mL LB, autoclaved) with 35uL of 100mg/mL Ampicilin and cells from WT Ascaris Hemoglobin glycerol stock in -80 freezer. | |||
#Shake/incubate the cultures overnight at 37C | |||
==Continuing Myoglobin Expression== | |||
# Spin down cells in starter culture (20 minutes, 4000 rpm, 4C) | |||
# Prep expression culture media | |||
## Add autoclaved phosphate to autoclaved TB with sterile technique. | |||
## Add 1mL of 100mg/ml Ampicilin | |||
# Innoculate expression cultures with resuspended cell pellets from starter cultures. | |||
# Shake at 165rpm and 37C until OD reaches 6.6 | |||
==Purification of WT Ascaris Hemoglobin== | |||
# Equilibrate Q column with water and then with Buffer A (25mM Tris, 50mM NaCl, pH 8) | |||
# Put sample on superloop and inject sample onto column | |||
## 200mL of sample. Adding to column will take longer than desired | |||
# Run 25mL of Buffer A over column after injection finishes | |||
# Start Eluting | |||
## Buffer B (25mM Tris, 1M NaCl, pH 8) | |||
## Gradient: 0-50% Buffer B in 30 minutes | |||
## Collect Fractions | |||
==Data== | ==Data== | ||
Revision as of 11:34, 27 June 2012
 Biomaterials Design Lab
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ObjectiveContinue sperm whale myoglobin expression as started by Tamra notebook page and finish purification of protein as started yesterday notebook page. DescriptionStart WT Hemoglobin Expression
Continuing Myoglobin Expression
Purification of WT Ascaris Hemoglobin
Data
NotesThis area is for any observations or conclusions that you would like to note.
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