Difference between revisions of "Team:UGA-Georgia/Experiments"
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</ol> | </ol> | ||
<p><sup>+</sup>Adapted from Qubit HS dsDNA</p> | <p><sup>+</sup>Adapted from Qubit HS dsDNA</p> | ||
+ | |||
+ | <h4>Making anaerobic formate media for <i>Methanococcus maripaludis</i> (400mL)</h4> | ||
+ | <ol> | ||
+ | <li>Glass-distilled water: 120mL</li> | ||
+ | <li>Glycylglycine buffer, 1M, pH=8.0: 80mL</li> | ||
+ | <li>General Salts Solution: 200mL</li> | ||
+ | <li>K<sub>2</sub>HPO<sub>4</sub> 14g/L: 4.0mL</li> | ||
+ | <li>Na acetate-3H<sub>2</sub>O, 136g/L: 4.0mL</li> | ||
+ | <li>Trace Mineral Solution: 4.0mL</li> | ||
+ | <li>Iron Stock Solution: 2.0mL</li> | ||
+ | <li>Sodium formate (NaCOOH): 10.8g</li> | ||
+ | <li>Sodium Bicarbonate (NaHCO<sub>3</sub>): 1.5g</li> | ||
+ | <li>Casamino acids: 0.8g</li> | ||
+ | <ul> Combine media ingredients and sparge with a stream of N<sub>2</sub> gas for >60minutes</ul> | ||
+ | <ul>Add 0.05g cysteine-HCl per 100mL and continue sparging for >10minutes.</ul> | ||
+ | <ul>Place in the anaerobic chamber overnight</ul> | ||
+ | <ul>Dispense media and pressurize to 15psi with N<sub>2</sub>/CO<sub>2</sub> gas</ul> | ||
+ | </ol> | ||
+ | |||
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Revision as of 23:26, 18 September 2015
Experiments
Transformation via PEG (1.5mL method - Plating and Enrichment)
- Check list of items on Day 1
- Day 1 (making plates)
- Day 2 (transformation and plating)
- Day 3 (enrichment in broth)
- Prepare Minispin and Vortex
- Preheat sandbox
- TB and TB+PEG Buffer
- Recipient strain of OD 0.7-1.0 in a rack (always make at least 2 days ahead)
- Selection markers
- Media and plates
- Latex gloves, tube locks, tips, pipette (1mL), microcentrifuge tubes
- Plasmid concentration determined (0.4-0.6ug per sample)
- 100mL beaker (for discarding supernatant)
- Melt plates (2 plates per sample) by autoclave, let cool down to 65C
- Add Na2S, antibiotics, and additives into plates and let solidify on side way
- Finish check list and prepare chamber (add H2 to 5.0, change catalyst and add CaCl2
- Wear latex gloves, get 2 1.5mL micro-tubes and place 1.5mL culture (OD 0.7-1.0) into each tube
- Centrifuge at 3,900rpm (Minispin) for 10 minutes
- Discard supernatant, add 75uL of TB, pipetting up and down for 10 times
- Add 0.2-0.3ug of plasmid and 45 uL of TB-PEG, pipetting up and down for 10 times
- Incubate at 37C in sand box for 1 hour
- While waiting, prepare 5mL of broth media plus Na2S in balch tube
- After incubation, combine 2 micro-tubes of cells into 1mL broth (total vol=1.24mL), vortex (strong)
- Take 2 new micro-tubes and add 0.9mL broth to each, now you have -2mL broth left in balch tube
- Do 100, 10-1, and 10-2 serial dilution with cells from the previous step, vortex (strong)
- Plate 0.6mL of cells from 100, and plate 1mL from 10-1 and 10-2. Lay plates sideways overnight before incubating at 37C
- Innoculate remaining cells (~0.6mL) from 10^0 to the balch tube with 2mL broth, record OD and incubate overnight at 37C
- Record OD again from the final step in Day 2, inoculate 0.5mL into 2 5mL of selective broth.
- Make frozen stocks of remaining.
Oxygen Exposure Protocol Pt.1 and Pt.2
- Check the ODs of the cultures (want ODs to be ~ 0.7)
- Relieve pressure inside the tubes.
- Remove the foil seal and rubber stopper
- Pour the 5mL contents in a 15mL tube with lid.
- Place in the rotor adapter and place adapter into rotor.
- Secure the rotor.
- Run a cycle at 3500rpm, for 20 minutes, at 4oC
- Then discard the supernatant of the cultures
- Re-suspend the cell pellets in 0.3mL of Pipes.
- Take the re-suspended cells to the sonicator.
- Centrifuge the product
- Collect the supernatant into the 0.5mL centrifuge tubes
- Microtip – 5(limit)
- Duty cycle – 50%
- Burst – 5sec.
- 20000G, 10 minutes, 4C
- Divide the remaining supernatant into ~5 60uL aliquots
- Place the aliquots into the freezer
Making a 1% agarose gel
- For 50mL gel: combine 50mL TAE 1X buffer and 0.5g agarose. For a 100mL gel: combine 100mL TAE 1X buffer and 1.0g agarose.
- Microwave the contents in a flask, with weighing paper covering the top, for 1.5minutes.
- Tape the sides of the gel mold and insert the well mold.
- Pour the contents into the gel mold.
- Allow to solidify (~20 minutes)
Sequencing Procedure*
- Determine which sequences warrant sequencing. The sequences that are sequenced need to have a strong Band around 900BP and a level of fluorescence greater than that of the negative
- Give the colonies new labels such as 1-10 that correspond with the a specific colony, For example L1C1A will be 1, L1C2A will be 2 and L1C1B will be 3.
- Spin PCR Product down
- Get 20ul and 200uL micro pipettes and tips
- Retrieve the DNA Clean&Concentrator-25(make sure it is 25 by checking size of columns)
- In a 1.5mL microcentrifuge tube, add 5 volumes of DNA binding buffer to 1 volume PCR Product
- Transfer Mixture to a provided Zymo-Spin Column in a collection tube
- Centrifuge for 30 seconds at 17g Discard Flow-through (set centrifuge timer to 1 minute and end 30 seconds early)
- Add 200 uL DNA wash buffer to the column, Centrifuge for 30 seconds at 17G
- Repeat previous step
- Add 25 uL water directly to column matrix and incubate at room temperature at room temperature for 1 minute,
- Transfer column to 1.5 ml microcentrifuge tube and centrifuge for 30 seconds to elute the DNA
- Remove Column
*Adpated from Zymo Research
Determining Protein Concentration+
- For each sample and one additional sample combine 199uL Quibit buffer and 1 uL Quibet Reagent to create solution; For example if one were testing 1 PCR product they would combine 398uL Buffer and 2 uL Quibet Reagent to create solution
- Add 1 uL to DNA to 199 uL solution
- Turn on Fluorometer
- Choose High Sensitivity, No New Standards DO NOT use first value given
- Press calculate sample stock and then change units nanograms/microliter
- Record Values in Lab Notebook and microcentrifuge tubes
+Adapted from Qubit HS dsDNA
Making anaerobic formate media for Methanococcus maripaludis (400mL)
- Glass-distilled water: 120mL
- Glycylglycine buffer, 1M, pH=8.0: 80mL
- General Salts Solution: 200mL
- K2HPO4 14g/L: 4.0mL
- Na acetate-3H2O, 136g/L: 4.0mL
- Trace Mineral Solution: 4.0mL
- Iron Stock Solution: 2.0mL
- Sodium formate (NaCOOH): 10.8g
- Sodium Bicarbonate (NaHCO3): 1.5g
- Casamino acids: 0.8g
- Combine media ingredients and sparge with a stream of N2 gas for >60minutes
- Add 0.05g cysteine-HCl per 100mL and continue sparging for >10minutes.
- Place in the anaerobic chamber overnight
- Dispense media and pressurize to 15psi with N2/CO2 gas