Team:UGA-Georgia/Protocols

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PROJECT

WET LAB

HUMAN PRACTICES

TEAM

PCR

  1. Amplified mCherry gene with negative control group and positive control group
  2. Reaction Mixture (a master mix to be divided into 3 tubes; Mq water 10.2 uL X 4 = 40.8 uL, 2x Phusion polymerase HF buffer mix 12.5 uL x 4 = 50 uL, Gene 0.5 uL x 4 = 2 uL, Forward primer 0.9 uL X 4= 3.6 uL, Reverse primer 0.9 uL x 4 = 3.6 uL)
  3. Final Reaction Mixture = 96.4 uL with 24.1 uL in each tube
  4. Annealing temperature = 65 Celsius
  5. Run products on a gel for verification (2.5 uL of each)

Plasmid Extraction

  1. Add 1000 uL of culture to each centrifuge tube
  2. Centrifuge tubes at 15000 rpm for 30 seconds
  3. Dispose supernatant in biohazard bin
  4. Add another 1000 uL of each culture to respective centrifuge tubes
  5. Centrifuge again at 15000 rpm for 30 seconds
  6. Discard supernatant in biohazard bin
  7. Add 600 uL of DI water to each centrifuge tube
  8. Vortex tubes to resuspend the pellet into the water
  9. Add 100 uL of 7x lysis buffer to each tube and mix for 30 seconds
  10. Add 350 uL of neutralization buffer and mix
  11. Centrifuge at 15000 rpm for 3 minutes
  12. Transfer supernatant to spin column
  13. Centrifuge at 15000 rpm for 30 seconds
  14. Discard liquid in collection tube
  15. Add 200 uL of endo wash buffer to each spin column
  16. Centrifuge at 15000 rpm for 30 seconds
  17. Add 400 uL of zippy wash buffer to each spin column
  18. Centrifuge at 15000 rpm for 1 minute
  19. Place spin columns in centrifuge tubes
  20. Add 40 uL of DI water to each column
  21. Let stand for 2 minutes (to allow the gene and plasmid to dissolve in the water)
  22. Centrifuge at 15000 rpm for 30 seconds
  23. Store at -20 Celsius
  24. Dispose of spin columns

Restriction Digestion

  1. PCR purified product, plasmid to be digested separately - choose the restriction enzymes and the buffer
  2. Set up: 24 uL of water, 5 uL of 10X buffer, 5 uL of 10X BSA, 2 uL of Restriction Enzyme 1, 2 uL of Restriction Enzyme 2, 12 uL of Vector/DNA
  3. Incubate at 37 Celsius for 2-4 hours in water bath

Gel Extraction

  1. Add 2 uL of DNA ladder and 50 uL of digested vector with 10 uL loading buffer dye into gel
  2. Run gel at 100 Volts until markers move half way down the gel
  3. Soak Gel in Ethidium Bromide for 10 minutes
  4. Observe gel under UV light
  5. Carefully cut out the part of gel which contain the vector and put into the centrifuge tube
  6. Add 200 uL of ADB buffer into each centrifuge tubes
  7. Place the centrifuge tubes in a 55 Celsius water bath for 20 minutes
  8. Shake well and place them back in water for 5 more minutes
  9. Pipet the solution from each tube into spin column-collection tubes
  10. Centrifuge at 8000 rpm for 1 minutes, and discard the supernatant
  11. Add 200 uL of DNA wash buffer to each spin basket
  12. Wait 2 minutes; then centrifuge at 15000 rpm for 30 seconds and discard the supernatant
  13. Transfer spin baskets to centrifuge tubes, and add 8 uL of water into each tube
  14. Let it sit for 2 minutes
  15. Centrifuge at 15000 rpm for 30 seconds
  16. Collect purified gene into one centrifuged tube

Ligation

  1. Made a solution of 60 uL of 2x buffer, 12 uL of digested vector, and 6 uL of quick ligase
  2. For each of the new centrifuge tubes(12 in total, properly labeled), add 3.5 uL of the previous PCR product and 6.5 uL of the solution into each tube in the order as labeled
  3. Let it sit for 10 minutes

Heat Shock Transformation

  1. Add 10 uL of the plasmid from ligation to each centrifuge tube, which contain 50uL of XL1-Blue cells, in the order as labeled
  2. Keep on ice for 30 minutes
  3. Heat shock at 42 Celsius for 60 seconds (6 tubes each time)
  4. Add LB, and keep on ice in 37 Celsius shaker for 45 minutes
  5. Centrifuge at 5000 rpm for 3 minutes
  6. Discard 600 uL supernatant
  7. Re-suspend the 100 uL culture
  8. Plate the 100 uL on each LB-ampicillin plate as labeled
  9. Place in the 37 Celsius Incubator

Formate Media

  1. Combine all the components of the media (900mL):
    • Glass-distilled water - 270mL
    • Glycylglycine buffer, 1M, pH=8.0 - 180mL
    • General Salts solution - 450mL
    • K2HPO4, 14g/L - 9.0mL
    • Na-acetate-3H2O, 136 g/L - 9.0mL
    • Trace Mineral solution - 9.0mL
    • Iron Stock solution - 4.5mL
    • Rezasurin - 0.9mL
    • Sodium formate (NaCOOH) - 24.3g
    • Sodium bicarbonate (NaHCO3) - 4.5g
    • Casamino acids - 1.8g
  2. Sparge with N2 gas for ~60minutes.
  3. Add 0.05g cysteine-HCl for every 100mL of media, then sparge for another 10 minutes.
  4. When sparging has been completed, but the media in the anaerobic chamber.

General Salts Solution

  1. Combine all the components:
    • KCL - 0.6g/L
      • medium concentraion - 4.5mM
    • MgCl2-6H2O - 5.50g/L
      • medium concentraion - 13.5mM
    • MgSO4-7H2O - 6.90g/L
      • medium concentraion - 14.0mM
    • NH4Cl - 1.00g/L
      • medium concentraion - 9.0mM
    • CaCl2-2H2O - 0.28g/L
      • medium concentraion - 0.95mM

Oxygen Exposure

  1. Take the ODs of all the cultures.
    • Make sure they are all above 0.6.
  2. Relieve the pressure from all the bottles/tubes.
  3. Remove the caps and seals from the bottles/tubes.
  4. Spin down the cultures to form "loose" cell pellets.
  5. When you have formed your cell pellet, immediately discard the supernatant.
  6. Re-suspend the cell pellets in 1.0mL of McM Media.
  7. Transfer the re-suspended cells into 1.5mL microcentrifuge tubes.
  8. Close the cap.
  9. Place the microcentrifuge tubes into a shaker at 37C for ~20hrs.
  10. Remove from the shaker and take to a plate reader to run fluorescence tests.

Components for McM Media

  1. dH2O - 50mL
  2. General Salts solution - 50mL
  3. K2HPO4, 14g/L - 1mL
  4. NaCl, 293g/L - 7.5mL

Separatory Funnel Extraction of Extracellular Content

  1. Take the ODs of all the cultures to be extracted.
    • These cultures should ideally be in the 0.5-0.9 range.
  2. Open the cultures using a de-clamp under the fume hood, relieving the culture of any pressure.
  3. Transfer the culture to a centrifuge ready container
  4. Centrifuge the cultures at 11,100G for 20 minutes, or until cell pellet is formed.
  5. After removing the samples from the centrifuge, carefully pour the supernatant into a separatory funnel, being sure to preserve all of the cell pellet. Place the cell pellet and its container into a -20C freezer for 1 hour, if planning on completing the extraction for cellular content.
  6. Pour 5ml of 9:1 hexane/acetone solution into the separatory funnel, there should be a distinct difference between the water phase layer and the organic phase layer.
  7. Carefully, yet thoroughly, mix the water phase and organic phase by inverting the separatory funnel, letting any built pressure escape by briefly removing the top to the separatory funnel after each inversion. Avoid letting any bubbles form by aggressive inversion.
  8. Allow the water phase and organic phase to settle until a distinct difference is visible again.
  9. Drain the supernatant into a container, leaving only the organic phase in the separatory funnel. This supernatant may be discarded.
  10. Drain all of the organic phase into a beaker and add 1.5g of sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into small clumps.
  11. Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary.
  12. Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample bottle.

Separatory Funnel Extraction of Intracellular Content

  1. Allow cell pellets to remain in -20C freezer for at least one hour, so cells may properly lyse.
  2. Resuspend cell pellet in 5ml 9:1 hexane/acetone solution. Use a micropipette to thoroughly mix cell pellet into hexane/acetone solution. Swirl this solution thoroughly for proper mixing.
  3. Pour this solution into a beaker and add 1.5g sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into clumps.
  4. Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary.
  5. Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample bottle.

Extraction of Organically Soluble Material from Methanococcus – Balch Tube/Sonication

  1. Take & record optical density (OD) values of cultures to be extracted. These cultures should ideally be in the 0.5-0.9 range.
  2. Open the cultures using a de-clamp under the fume hood, relieving the culture of any pressure with a needle, if necessary.
  3. Transfer the culture to a centrifuge-ready container.
  4. Centrifuge the cultures at 11,100G for 20 minutes, or until cell pellet is formed.
  5. After removing the samples from the centrifuge, carefully pour the supernatant into a balch tube, being sure to preserve all of the cell pellet.
  6. Prepare the cell pellet and complete sonication to lyse cells, if planning on completing the extraction for cellular content.
  7. [Either combine the supernatant & cell lysate, or extract them separately.]
  8. Measure out & pour 12-15ml of Methylene Dichloride (DCM) into the balch tube with the extract, there should be a distinct difference between the water phase layer and the organic phase layer. [DCM will be the bottom layer as DCM is denser than water.]
  9. Cap the balch tube, and mix the water phase and organic phase by rapidly vortexing for 60 seconds. Allow any built pressure to escape by briefly removing the cap during mixing.
  10. Allow the water phase and organic phase to settle until a distinct difference is visible again.
  11. Using a micropipette, carefully remove and discard the water phase, leaving only the organic phase in the balch tube.
  12. Pour all of the organic phase into a beaker and add 1.5g of sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into small clumps.
  13. Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary.
  14. Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample vial.

Anaerobic Transformation (1.5ml method - Plating & Enrichment)

  1. Check List of items on Day 1
    • Prepare Minispin & Vortex
    • Preheat sand box
    • TB and TB+PEG Buffer
    • Recipient strain of OD 0.7-1.0 in a rack (always make it at least two 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 dumping supernatant
  2. Day 1 (making plates)
    • Melt plates (2 plates per sample) by autoclave, let cool down to 65C
    • Add Na2S, antibiotics & 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
  3. Day 2 (transformation and plating)
    • Wear latex gloves, get TWO 1.5ml micro-tubes and place 1.5ml culture (OD 0.7-1.0) into each tube.
    • Centrifuge at 3,900 rpm (Minispin) for 10 minutes
    • Discard supernatant, add 1ml of TB, vortex (strong: 8th gear)
    • Centrifuge at 6,500 rpm for 10 minutes
    • Discard supernatant, add 75ul of TB, pipetting up & down for 10 times
    • Add 0.2-0.3ug of plasmid and 45ul of TB-PEG, pipetting up & 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 10^0, 10^-1 and 10^-2 serial dilution with cells from the previous step, vortex (strong)
    • Plate 0.6ml of cells from each dilution, put plates on side way overnight before incubating at 37C
    • Innoculate remaining cells (~0.6ml) from 10^0 to the balch tube with 2ml broth, record OD & incubate overnight at 37C
  4. Day 3 (enrichment in broth)
    • Record OD again from the final step in Day 2, inoculate 0.5ml to 5ml of selective broth

Plate Reader

  1. We used the Gen5 Software.
  2. We created a procedure to read both in the spectrum of mCherry and within the auto-flourescence spectrum.
  3. Allow the machine to warm up.
  4. Run a blank plate.
  5. Fill the individual wells of your choosing (96 total) with a maximum of 100uL.
  6. Check all wells that you want to be read in the program.
  7. Run the trail.

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