Difference between revisions of "Team:Virginia/Experiments"
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Revision as of 05:36, 1 November 2017
Protocols
Pc. denitrificans Protocols
- Grow Pc. denitrificans cultures in liquid media
- Dilute 500mL of Pc. denitrificans culture in 500mL SGM17 medium
- ***Cold Room from here on***
- Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. carefully dump out supernatant
- Completely resuspend the pellets(shake side to side to avoid liquid loss) in all centrifuge bottles each with 50mL of 0.5 M sucrose solution with 10% glycerol
- Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add appropriate amount of water to the other empty centrifuge bottle for counterbalance
- Centrifuge the two bottles at 5000xg at 4 C
- Dump out supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water
- Centrifuge the two bottles at 5000xg at 4 C
- Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
- Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes
- Flash freeze with liquid nitrogen and store at -80 C
- Thaw and mix 50 uL portions cells with 5 uL DNA
- Transfer to ice cold electroporation cuvette
- Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
- The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
- IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
- Dilutions were made in the SGM17 media
- Incubate cells at 30 C for 2 hours
- Take 100 uL portions and plate.
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Pc. denitrificans Nutrient Agar
- Combine 23g 3 Nutrient Agar/Broth Agar Medium Nutrient Agar with 1000 mL deionized water, then autoclave
- 3g Nutrient Agar Composition Beef Extract, 5g peptone, 15g agar
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Pc. denitrificans Nutrient Broth
- Combine 8g Medium Nutrient Broth with 1000 mL deionized water, pH to 6.8 +/- 0.2, then autoclave
- 3g Nutrient Agar Composition Beef Extract, 5g peptone
Measurement Protocols
-
Isolate Single colonies
- Select glycerol stock cultures from -80 C storage to begin
- Thaw on ice for 15 minutes and observe when the cell stock begins to melt
- Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
- If the culture is Pc. denitrificans, culture on PD media or standard LB
- If the culture is E. coli use LB media
- If using untransformed bacteria, equivalent non-selective media should be used
- Incubate both Pc. denitrificans and E. coli overnight at 37 C
- Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics
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Preculture for Device Testing
- Allow the resuspended liquid culture to incubate for 4 to 6 hours in a shaking incubator at 37 C and 220 rpm to reach late- to mid-exponential phase of growth, look for an OD 600 value of ~0.6
- Obtain a 1 mL sample and measure the OD600. If at a satisfactory level, then proceed with device testing
- Divide the 60 mL pre-cultures into 3.5 mL aliquots in 14 mL falcon RB tubes or comparable sterile culture tube
- Prepare one tube for each hour that the planned test calls for, mark each tube with the number of hours it will remain unsealed and permeable to oxygen
- At the start of testing, cover the tubes marked “0 hours” with parafilm and leave sealed for the duration of testing
- Start incubation in a shaking incubator at 37 C and 220 rpm
- Every hour stop the shaking incubator and cover the next tube with parafilm. Repeat this step until all tubes except the last ones are covered
- Allow the final set of tubes to incubate unsealed for an hour and stop the incubation
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Device Measurements
- Starting with the unsealed tube, use a vernier Dissolved Oxygen (DO) membrane probe or comparable measurement system to measure the do levels
- Do this for all samples that were left open for a given time interval before moving to the next
- In between DO measurements, clean the probe with ethanol and then rinse with deionized water to minimize cross-contamination between samples
- Immediately take three 150 uL samples of the culture and dispense into appropriate wells in an optical PCR well plate
- Take a 500 uL sample and dilute with 500 uL deionized water for a total volume of 1 mL in a spectrophotometer cuvette to make a final 1:2 dilution. Store on ice if possible.
- Once all tubes have been unsealed and measured for DO content, seal the PCR optical well plate with an optical grade adhesive cover
- After all samples for optical fluorescence measurement have been taken, remove cuvettes from storage to measure and record OD600 absorbance values
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PCR Fluorescence Measurement
- Immediately after all samples have been collected and the well plate covered, run a pre-created protocol for fluorescence measurement
- The protocol should have three measurement cycles set at 30 seconds and 37 C
- After measurements are made by the optical reader, export the data for analysis
- f the Bio Rad MyiQ Optical Reader is used, copy and paste the relative fluorescence unit table presented by the output and export to either a .txt or .excel format
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Disposal
- Disinfect all Cuvettes with bleach or ethanol before washing with tap/sink water. Then rinse with deionized water and wipe dry
- All remaining tubes should be sterilized with bleach or ethanol and drained down an appropriate lab sink. All tubes are to be disposed of in a lab waste receptacle
- All PCR well plates should be sterilized disposes of similarly
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