Team:WPI Worcester/Experiments

LB Media

Material

  • 2 Liter Flask
  • LB Broth Powder
  • DI Water
  • Bottles
  • Stir Bar
  • 1 Liter Graduated Cylinder
  • Balance
  • Cup

Protocol

  1. Place a large stir bar in a 2 liter flask
  2. Place the 2 liter flask on a stir plate
  3. Use a 1 liter graduated cylinder to measure 1 liter of DI water and pour it into the 2 liter flask
  4. Turn on the stir plate
  5. Place a cup on a balance and tare the balance
  6. Use a scoop to measure out 40 grams of LB Broth powder
  7. Carefully add the 40g of LB broth powder to the 2 liter flask
  8. Allow to mix until all of the powder is dissolved
  9. Pour The LB into smaller (250mL) bottles
  10. Leave caps slightly loose and tape with autoclave tape
  11. Autoclave the LB
  12. Tighten caps

LB Agar

Materials

  • 2 Liter Flask
  • Stir plate
  • Petri Dishes
  • 50℃ Water Bath
  • Bunsen Burner
  • Paper Towel
  • Colored Sharpie
  • Tinfoil
  • DI Water
  • Balance
  • Cup
  • Stir bar

Protocol

  1. Tare the balance with a cup on it
  2. Measure 40 grams of LB Agar into the cup
  3. Put 1 liter of water in a 2 liter flask on a stir plate
  4. Add the LB agar into the flask and a stir bar into the flask
  5. Allow to mix until powder is dissolved
  6. Put tinfoil on the top of the flask and add a strip of autoclave tape
  7. Autoclave the flask
  8. Mix the flask again
  9. Place the flask in 50℃ water bath for 30 minutes to 2 hours
  10. Add antibiotic to the flask (1mL to 1L)
  11. Prep empty petri dishes in stack of 5 for easy pouring
  12. Label petri dishes with appropriate color that corresponds to antibiotic
  13. When ready to start pouring, light the bunsen burner and retrieve the flask from the water bath
  14. Wrap a paper towel around the neck of the flask to avoid it getting slippery while pouring
  15. Before pouring each stack of 5 petri dishes, place the lip of the flask into the flame of the bunsen burner to ensure it is sterile
  16. Pour all of the plates
  17. Let the plates cool overnight
  18. Bag the plates and store in the fridge for up to a few months

DNA Miniprep

Materials

  • A1 buffer
  • A2 buffer
  • A3 buffer
  • A4 buffer
  • Elution buffer
  • Overnight culture of E. Coli
  • Column
  • Microfuge tube

Protocol

  1. Prepare an overnight culture of the E. coli with desired plasmid (5mL LB + 1 colony + 5uL of desired antibiotic)
  2. Incubate in shaker at 37℃ overnight
  3. Spin down culture in big centrifuge at 3500 rpm for 10 minutes
  4. Dump out supernatant into waste beaker
  5. Resuspend pellet in 250 uL A1 buffer
  6. Move culture into 1.5mL microfuge tube
  7. Add 250 uL A2 buffer
  8. Incubate for 5 minutes (mix well, should be blue and sticky)
  9. Add 300uL of A3 buffer (mix until white and fluffy)
  10. Spin in small centrifuge at max speed for 10 minutes
  11. Set up and label columns while sample is spinning
  12. Move supernatant into the column
  13. Spin in small centrifuge 30 seconds max speed
  14. Dump liquid
  15. Add 600uL of A4
  16. Spin for 30 seconds at max speed
  17. Dump liquid
  18. Spin for 2 minutes
  19. Move column into microfuge tube
  20. Add 50uL elution buffer
  21. Spin 1 minute at max speed
  22. Nanodrop to check yield
  23. Plug in the nanodrop
  24. Wash the metal plates with DI water
  25. Dry with kimwipe
  26. Put 2uL elution buffer onto bottom plate
  27. Run blank
  28. Confirm blank by running elution buffer again
  29. Wipe off plates and then run samples
  30. 2uL of sample and record the ng/uL

Transformations

Materials

  • Competent cells
  • 1.5mL microfuge tubes
  • Plasmid DNA
  • Ice bucket
  • Water Bath (42℃)
  • SOC Medium
  • 37℃ shaker
  • Agar plates
  • Incubator

Protocol

  1. Thaw competent cells on ice (~30 minutes)
  2. Pre-chill 1.5mL microfuge tubes
  3. Add 50 uL of competent cells into each microfuge tube
  4. Add 1uL of plasmid DNA into the tubes
  5. Incubate on ice for 30 minutes
  6. Heat shock in a water bath (42℃) for 45 seconds
  7. Incubate on ice for 5 minutes
  8. Add 250uL of SOC medium to each tube
  9. Incubate in shaker at 37℃ for 1 hour
  10. Plate the 250uL of cells onto agar plates
  11. Incubate overnight

NCTC Electroporation

  1. Autoclave 500mL of LB in a 1 liter flask
  2. Autoclave ~2000mL of DI water (then put in 4℃ fridge)
  3. Make 50mL of overnight culture
  4. Let incubate overnight
  5. Add the 50mL overnight culture to 500mL LB in flask
  6. Put in 37℃ shaker until the OD is at ~0.4
  7. Centrifuge in big centrifuge at 3500rpm for 10 minutes at 4℃
  8. Discard supernatant (dump into waste and tap on paper towel)
  9. Resuspend in 500mL of ice cold ddH2O and spin and dump
  10. Do this rinse three times at least
  11. Resuspend in 1mL Ice cold 10% glycerol
  12. Make 100uL aliquots (Keep in -80℃ freezer or on ice if electroporation is happening directly after)
  13. Chill 1.5mL microfuge tubes
  14. Add 50uL of cells to the microfuge tube
  15. Add 1uL of plasmid DNA to the microfuge tube
  16. Incubate on ice for 10-30 minutes
  17. Carefully put into the electroporation cuvette
  18. Get volume to 100uL using ddH2O
  19. Put cuvette in electroporation apparatus
  20. 2-5 kilovolts
  21. Add 950uL SOC medium to the electroporation cuvette to rinse the cells out of cuvette
  22. Move cells into 15 mL tube and incubate for 1 hr at 37℃
  23. Plate the cells on agar plates with appropriate antibiotic

M9

M9 Salts

  1. 800mL H2O
  2. 64g Na2HPO4-7H2O
  3. 15g KH2PO4
  4. 2.5g NaCl
  5. 5g NH4Cl
  6. Stir until dissolved
  7. Adjust to 1000mL with distilled H2O
  8. Separate into 5 200mL aliquots
  9. Autoclave to sterilize

M9 Media

  1. 700mL ddH2O
  2. 200mL M9 Salts (above)
  3. 2mL MgSo4 (sterile)
  4. 20mL of 20% glucose (or 20% glycerol)
  5. 100uL of 1M CaCl2
  6. 1mL of sterile, filtered 1000x L-Arginine
  7. Adjust to 1000mL using ddH2O

Mg Chemically Competent Cells

Materials

  • 15mL conical tubes
  • 1.5 mL microfuge tubes
  • CCMB80 buffer
  • Overnight culture
  • Centrifuge

Protocol

  1. Prepare an overnight culture (x4) (5mL LB + 1 colony)
  2. Incubate overnight
  3. Spin overnight culture for 10 minutes at 3500rpm
  4. Pour supernatant into waste beaker
  5. Resuspend in 80mL of CCMB80 buffer (20 in each tube)
  6. Incubate on ice for 20 minutes
  7. Spin for 10 minutes at 3500rpm
  8. Resuspend in 10mL CCMB80 buffer (2.5 in each tube)
  9. Combine cells from all tubes
  10. Incubate on ice for 20 minutes
  11. Aliquot 400uL into each microfuge tube
  12. Store at -80℃ and discard thawed cells

Protein Purification

Materials

  • Lysis Buffer
  • Equilibration Buffer
  • Wash Buffer
  • Elution Buffer
  • BL21 Cultures of GFP, IA, and ZeAFP
  • Tissue Grinder
  • NiNTA Slurry

Protocol

  1. Add 5mL of LB, 5uL of AMP, and one picked colony from the BL21 strains and grown overnight
  2. Colonies grown overnight
  3. 1 mL of culture were added to 200mL of LB
  4. OD was checked. Goal was to be between 0.05-0.1 before incubation
  5. After incubation for 3 hours, the move the cultures to 50 mL tubes and spin cultures for 10 minutes
  6. Remove the supernatant and freeze the pellets. Each bacterial strain had 5 conical tubes for a total of 25 tubes total
  7. Combine all pellets into 5mL of lysis buffer (1mL of lysis buffer into each conical tube)
  8. Combine all 5 tubes of the same protein into one labeled 15 mL conical tube
  9. Incubate on ice for 20 minutes
  10. Dounce each 5mL sample on ice 40 times (1 down and 1 up equals 1 time)
  11. Clean tissue grinder with water and rinse with lysis buffer between each sample
  12. Pellet 4℃ at 10,000 rp, for 15 minutes
  13. Prepare the Ni∙NTA Agarose
  14. Add 0.5mL Ni∙NTA slurry into a tube. Mix the solution well before pipetting
  15. Add 2mL of equilibration buffer per tube to wash
  16. Wash 2 times with Equilibration buffer, and drain the buffer between washes
  17. Completely drain buffer after the last wash
  18. Take the supernatant out of the tubes in the centrifuge and add each to a labeled Ni∙NTA Agarose tube.
  19. Rotate the samples at 4℃ for 1 hour-overnight. Remove 100 uL of the input sample and store at -80℃
  20. Spin tubes to collect the Ni∙NTA
  21. Supernatant sample was taken (100 uL at -80℃)
  22. Use 2mL of wash buffer on ice 3 times. Centrifuge between washes for 1 minute at 1,000g to collect supernatant
  23. Drain the final wash completely
  24. Put 1 mL of elution buffer into the tubes
  25. Tip back and forth every minute for 5 minutes on ice
  26. Centrifuge all
  27. Remove supernatant and collect completely
  28. Store all at -80℃.

SDS Page Gel

Protocol

  1. Add 10 uL of input, 10 uL of supernatant, 2 uL of eluate and 2 uL of agarose to eppendorf tubes. Add 10 uL of buffer to each tube
  2. Spin tubes for 1 minute and put in an 80℃ water bath for 10-20 minutes
  3. Spin tubes for another minute
  4. Load 20 uL of the ladders, input and supernatant into their specific well
  5. Load 12 uL of the eluate and agarose to their specific wells
  6. Run the gel for 1.5hrs
  7. Remove gels, cover with water, and wash 3 times.
  8. Add dye to cover the gel for an hour
  9. Rinse gel with water
  10. Add water to cover the gel and shake overnight
  11. Rinse off dye and read gel

Cloning Protocol

PCR Clean Up

  1. Take 45μl of PCR reaction (ie. Lp , Cf , Dc) and add it to 90μl of NT1 buffer and add both to a column. Place the column in a tube.
  2. Spin at max rpm (approximately 1300) for 1 minute.
  3. Empty out the tube and add it back to the column. Add 700μl of NT3 buffer.
  4. Spin at max rpm for 1 minute.
  5. Empty out the tube and add it back to the column. Spin at max rpm for 1 minute.
  6. Empty out the tube and dispose of it. Place column in a microfuge tube.
  7. Add 40μl of elution buffer. Incubate at room temperature for 1 minute.
  8. Spin at max rpm for 1 minute.
  9. Dispose of the column.
  10. Determine the DNA concentration using a nanodrop device (in ng/μl)
  11. Place the microfuge tube containing purified DNA in -20oC storage for later use.

Gibson's Assembly

  1. Each Gibson Assembly mixture is comprised of multiple components. Total volume for each tube in 10μl, with 1μl being vector the volume of DNA and water being dependent on the DNA concentration. Spin all tubes down before any volumes are added.
  2. 10μl of 2x Gibson Assembly master max is added to each tube.
  3. Incubate each tube for 1 hour at 50oC.

Transformation

  1. Take 2μl of each Gibson Assembly reaction, placing any left over reaction in the -20oC and add it to 50μl of cells (NEB 5 alpha super-competent cells).
  2. Place each tube on ice for 30 minutes.
  3. Place each tube in a 42oC water bath for exactly 30s.
  4. Place each on ice for 5 minutes.
  5. Add 950μl of SOC recovery medium to each tube. The total volume of each tube should be approximately 1000μl.
  6. Incubate at 37oC for 2 hours.
  7. Plate each tube on ampicillin-resistant plates in two volumes, 100μl and 900μl, for a total of 10 plates.
  8. Check the plates the next day for colonies.

Crystal Violet Biofilm Assay

Materials

  • 100 mL M9 media (CONCENTRATION at 1000x) and glutamine (CONCENTRATION at 100x), filtered through a 0.2µm vacuum filtration unit once at 100mL)
  • 51 mL LB media (no antibiotics present)
  • agar plates containing streaked bacteria of desired strains (DH5α, EMG-2Kλ, NCTC 9001, S. epidermidis, S. aureus)
  • 5 50mL conical tubes
  • 11 15mL conical tubes
  • 4 U-bottom 96 well tissue culture plates
  • 2 flat bottom 96 well tissue culture plates
  • 0.1% crystal violet (0.2g crystal violet in 200mL water)
  • 33.6 mL 30% acetic acid

Protocol

Note: steps 4-9 should be done within the sterile field of an ethanol-cleaned lab bench under a Bunsen burner to minimize contamination.

  1. Add 10 mL LB in 50 mL tubes
  2. Pick one colony from each plate (DH5α, EMG-2Kλ, NCTC 9001, S. epidermidis, S. aureus) and put one in each 50 mL tube.
  3. Tighten caps then loosen a half turn. Tape the cap on, then incubate in the shaker overnight at 37°C and 175 RPM. CHECK SPECIFICATIONS
  4. Dilute all cultures except NCTC 9001 1:20 (0.5mL culture, 9.5 mL M9) in 15mL conical tubes. Dilute NCTC 9001 1:10 (1mL culture, 9mL M9).
  5. Check OD600 of each culture, using a blank of 1:20 LB in M9. (0.25mL LB in 4.75mL M9 is sufficient.)
  6. Adjust OD600 to 0.1 using M9 for a total of 10mL, combining diluted culture and M9 into 5 new conical tubes.
  7. Check OD600 again to verify it is 0.1.
  8. Pipet 100μL of the OD600= 0.1 solution of DH5α into each of the first 6 wells of the first row of each of the four plates. Repeat with the next four strains and the next four rows.
  9. Pipet 100μL of M9 into the first 6 wells of the sixth row of each of the four plates.
  10. Set two of the plates in the incubator at 37°C. Leave the other two on the lab bench.
  11. After 48 hours, take one plate from the incubator and one plate from the bench.
  12. Rinse each plate two to three times with distilled water.
  13. Pipet 125μL of 0.1% crystal violet into each well previously containing liquid (rows A-F, columns 1-6 of both plates).
  14. After 10 minutes, rinse both plates with distilled water three to four times.
  15. Pipet 200μL of 30% acetic acid into each well previously containing liquid (rows A-F, columns 1-6 of both plates).
  16. Pipet up and down four times then transfer 100mL of each well from the room temperature plate into the corresponding well in a flat bottom 96 well plate. Repeat for the 37°C plate, but transfer into the empty wells (A7 - F12) on the right side of the same flat bottom 96 well plate.
  17. Pipet 100μL of 30% acetic acid into each well of row G of the flat bottom 96 well plate.
  18. Process OD595 in an absorbance plate reader.
  19. After 24 hours, repeat steps 12 - 18 with the remaining plates.

Coated Protein Trials

Growing Cells

  1. Overnight cultures of S. Epi, S. Aureus, EMG, DH5Alpha, and NCTC were grown in 5 mL of LB (0.5 mL of these were then added into 9.5mL of M9 minimal media and diluted to be between 0.05 and 0.1 (OD600))

Protein Coated Plates

  1. 25 uL of protein, GFP, IA4, IA5, Ze4, and Ze5, were added to each well
  2. These dried for 3 hours and any excess protein was removed
  3. All plates were put in the incubator for 48 hours at 37 degrees C

Mixed Protein Plates

  1. 100 uL of bacteria were added into each well according to the charts above. On plates 1-4, 10uL of protein were added to specified wells
  2. All plates were put in the incubator for 48 hours at 37 degrees C

The crystal violet procedure was followed from this point on

Gold Particle Preparation for the Gene Gun

Biolistics PDS-1000/He Particle Delivery System