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Creating a E.Coli Stock Culture
<!--''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Requesting Balance Calibration]]
*[[Requesting Pipette Calibration]]
*[[Using the UV-Vis]]
*[[Using and Maintaining pH Meter]]
*[[-80 Freezer Storage and Maintenance]]
*[[Freeze Dryer Maintenance]]
*[[Lab Notebook Guidelines]]
*[[Sample Barcoding]]


1. Make LB media as per the protocol titled Media Preparation
''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]


2. Transfer 25mL of LB media in to a sterile (autoclave) 250 mL  erlenmeyer flask using a sterile Falcon Transfer Pipet
''Data Collection''
*[[Gap Sampling]]
*[[Water Suppression with presaturation pulses (zgpr/zgcppr]]
*[[Non-uniform Sampling]]
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]


3. Inoculate the flask with enough stock culture(Glycerol stock obtained from a lab mate/purchased) to create a final OD600 of 0.025
''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
*[[Non-Uniform Sampling]]
*[[Chara]]
*[[2D NMR Processing in Linux and Windows]]
*[[2D Metabolomics NMRPipe Processing]]
*[[1D NMR Processing in Linux and Windows Example Script]]
*[[NMR Batch Correction Example Script]]
*[[PCA Classify Example Script]]
*[[Sample Collection and Processing for Protein Backbone Assignments]]
*[[Example Scripts for NMRPipe Processing]]


4. Make note to have 1:10 ratio of liquid to air in your inoculated flask for aerobic growth of E.coli


5. Incubate the flask at 37C and a shaker speed of 100-150 rpm
''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]
*[[Setting Up & Running MD Simulations]]
*[[Analyzing MD Simulations]]
*[[iRed Analysis of MD Simulations]]


6. Over every 30 min take an OD600 reading to track the growth of the E.coli cells in your flask
Cell Culturing
*[[Cell Culturing Dr. Franco Lab]]


7. When the cells hit Stationary phase (OD600 greater than or equal to 10 in 1ml of culture) the culture is ready to make stock tubes for storage
''Metabolomics''
*[[Metabolite Extraction]]
*[[MetPa for metabolomics]]
*[[Making Heatmaps]]
*[[One way ANOVA in R]]
*[[P-Value adjustment for multiple comparisons]]
*[[PCA-Utils]]
*[[NMR Tube Deep Cleaning]]
*[[Noise Removal for PCA]]
*[[Weighted Linear Least Squares]]
*[[Serum Preparation for 1D NMR]]
*[[Whole Blood Preparation for 1D NMR]]
*[[Urine Preparation for 1D NMR]]
*[[Cell Culture Preparation for 1D NMR]]
*[[LC-MS metabolomics guide-CIBC collaborations]]


8. At this point, take 1ml of culture and add 0.5ml of glycerol in a 2ml sterile tube, create 10-15 of these 2ml glycerol stock tubes


9. Store the tubes with appropriate labeling in the -80C freezer for until you need to use them again.
''Demos''
*[[Peroxide Clock]]
*[[Traffic Light Reaction]]
*[[Orange Juice Clock]]
*[[Gummy Bear Freeze]]
*[[Lava Lamps]]
*[[Women in Science: Checklist]]
*[[Maxey Day: Checklist]]
-->

Latest revision as of 04:31, 11 September 2021

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