MiniBsubProcessProcedures

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Process Development for Reduced Genome Project - draft of standard operating procedures

I. Synchronization of Activities
II. Pre-process
	A. Media preparation
	B. PCR primer generation
	C. PCR joining of primer to antibiotic resistant gene cassette
	D. freezing and thawing strains
III. Process
	A. Culturing requirements for B.subtilis on plates (partial pressure of O2 & CO2, pH, temp ...)
	B. Antibiotic selection compounds & ISO suggestions for working with them
	C. Cell yield of reduced strain (what is a success / how many do we want to keep ...)
IV. Validation
	A. Reduced media studies
	B. Sequencing over seam
	C. Fermentation studies (for scale up)

Related Information
	I. Equipment List and Procurement Information

1 '''Putative Essential Genes in B subtilis'''

1.1 '''competence proteins'''

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2 Lab Supplies

• Scales
• Microscopes (brightfield and phase contrast?)
• Refrigerator
• Water bath/shaker
• Heat block
• Stirrer
• Pipetters
• Petri dishes
• Label maker
• Sharpies/china markers
• Burners
• Stain (slide preparation)
• Filters (gross and sterilization)
• Pipette tips
• Wipes
• Paraffin sheets
• Tin foil
• Acetone (or other surface sterilizer)
• Hand wash
• Scissors, knives, razor blades
• Syringes
• Swabs
• Innoculation loops
• Safety goggles
• Lens cleaner

Glassware (does UChicago have a store?)

• Beakers
• Flasks
• Stirring rods
• Plate spreaders
• Tubes
• Pipettes
• Slides

Major equipment (can we use some other lab's stuff?)

• Incubator (culture and hybridization?)
• Centrifuge (multi-speed or multiple units?)
• Autoclave
• Anaerobic chamber/kits
• Densometer
• Spectrometer
• Microarraying
• PCR
• Thermal cycler
• Agarose (and/or polyacrylamide?) gels
• Electrophoresis (protein and/or DNA/RNA?)
• Vertical (SDS-PAGE)? 2-D? Analytical?
• Power supply
• Electrodes
• Cells
• Combs
• Casting kits
• Gel dryer
• Precast gels (?)
• Buffer solutions, reagents
• Stains
• Markers/dyes

Biological supply

• Strains
• Media (liquid, solid, additives) -- prepared or dried?
• Antibiotic discs (and/or other delivery methods?)
• Primers (DNA/RNA)

Misc notes

• Who does UChicago use for instrument calibration?
• Can they identify biological/lab supply places for us?
• Is it possible to use a lab set-up service/consultancy? (These probably cost a fortune.)
• Do the labs already have a functional distilled water supply? Fume hoods working?
• What other reference materials are needed?
• Some second-hand lab equipment places:
• http://www.labrecyclers.com (partner with http://www.rapidlabsolutions.com)
• http://www.amebioscience.com (they're based in Europe, though)
• http://www.biosurplus.com