Team:MichiganState/Protocols

Protocols

Media Solution Preparation
"P" Media

Media Protocol for 50 mL solution:

  1. Under sterile conditions, add 5 mL of the 10x Solution 1
  2. Add 1.5 g NaCl
  3. Add 50 uL of trace solution
  4. Add milliQ water to 50 mL
  5. Autoclave for 30 minutes
  6. Once solution is cool, add 2.5 mL of Phosphate Solution
  7. Add 2 mL carbonate solution, close and store at room temp

Trace Solution

Na2EDTA x 2H2O 5 (g/L)
FeSO4 x 7H2O 2
ZnSO4 x 7H2O 0.3
MnCl2 x 4H2O 0.03
CoCl2 x 6H2O 0.2
CuSO4x 1.2H2O 1.2
Na2O4W x 2H2O 0.3
NiCl2 x 6H2O 0.05
Na2MoO4 x 2H2O 0.05
H3BO3 0.03

Phosphate Solution

KH2PO4 5.44 (g/L)
Na2 HPO4 5.68

Carbonate Solution

NaHCO3 75.6 (g/L)
Na2 CO3 10.5

Solution 1

KNO3 1 (g/L)
MgSO4 x 7H2O 0.2
CaCl2 x 2H2O 0.02
"MP"/"MPM" Media

Media Protocol for 150 mL solution:

  1. Under sterile conditions, add 7.5 mL of the Pipes (20x) solution
  2. Add 1.5 mL of P-Sol
  3. Add 600 uL of (NH4)SO4
  4. Add 37.5 uL of MgCl2
  5. Add sterile water up to the 150 mL line
  6. Add 150 uL of C7
  7. Add 1.5 uL of CaCl2
  8. Close and store

To Make "MPM" Media

  1. Add 750 uL of methanol, close and store

    2. NOTE: MP Media will last for a few months. To maintain precise results, make MPM when needed as methanol concentration will decrease over time.

Note: Pipes used CAS: 5625-37-6

P Solution

KNO3 1 (g/L)
MgSO4 x 7H2O 0.2
CaCl2 x 2H2O 0.02
Extractions
Plasmid Extraction
  1. Grow strain with pAWP78 on LB/Kan plate overnight
  2. Inoculate 5 mL of LB/Kan broth with plasmid-containing strain and shake at 200 rpm at 30 ºC
  3. Take cultures from shaker, should be opaque
  4. Add 1.5 mL from culture and put into an Eppendorf tube
  5. Centrifuge for 1 minute at 13,000 rpm, room temperature
  6. Decant excess media in liquid waste
  7. Repeat steps 4-6 two more times in the same Eppendorf tube
  8. Use a 100 uL pipette set at 50 uL to remove excess media, but do not touch the pellet
  9. Using materials from the GeneJET Plasmid Miniprep Kit, obtain 250 uL of resuspension solution and resuspend the pellet
  10. Add 250 uL of lysis solution and invert 2-3 times to mix
  11. Add 350 uL of Neutralization solution and invert 3-4 times
  12. Centrifuge for 5 minutes at 13,000 rpm
  13. Transfer the solution to a purification column and centrifuge at 13,000 rpm for 1 minute
  14. Discard liquid that passes through and add 500 uL of Washing Solution
  15. Centrifuge for 1 minute at 13,000 rpm and discard liquid that passes through
  16. Wash again and spin down again and discard the solution
  17. Spin down the tube again with nothing in it for 1 minute at 13,000 rpm
  18. Add 30 uL of milliQ water and incubate at room temperature for 2 minutes
  19. Replace bottom tube with a clean Eppendorf tube and centrifuge for 2 minutes at 13,000 rpm
  20. Discard column and use the solution in the Eppendorf tube to analyze it on a nanodrop
Gel Extraction
  1. Weigh a 1.5 mL microcentrifuge tube
  2. Cut desired fragment out of the gel and add to the test tube
  3. Weigh again and calculate weight of gel
  4. For every 100mg of Agarose gel add 200uL of Buffer NTI
  5. Incubate the sample for 5-10 minutes at 50ºC
  6. Vortex the sample briefly every 2-3 minutes until completely dissolved during incubation
  7. Place a Nucleospin Gel and PCR Clean-up column into a collection tube (2mL) and load up to 700uL of sample
  8. Centrifuge for 30 seconds at 11,000 x g
  9. Discard the flowthrough and place the column back in the collection tube
  10. Load the remaining sample if necessary and repeat spin-down
  11. Add 700uL of Buffer NT3
  12. Centrifuge for 30 seconds at 11,000 x g and discard the flowthrough
  13. Repeat steps 11 and 12
  14. Centrifuge for 1 minute at 11,000 x g and discard the flow through
  15. Incubate for 2-5 minutes at 70ºC to get rid of ethanol
  16. Place the spin-column in a new 1.5mL microcentrifuge tube
  17. Add 30uL of Buffer NE and incubate at room temperature for 1 minute
  18. Centrifuge for 1 minute at 11,000 g
1% Agarose Gel Electrophoresis
  1. Measure 1.25 g agarose
  2. Mix agarose with 125mL of SB buffer
  3. Microwave until dissolved
  4. Wait until it cools to about 50 degrees Celsius
  5. Add 1 uL of Labsafe Nucleic Acid Stain for every 10mL of gel
  6. Mix and pour into a gel casting tray
  7. Add combs and allow it to solidify
  8. After gel has solidified cover gel with SB (sodium borate) buffer and remove combs
  9. Mix 2 uL of DNA ladder with 1 uL of loading dye and pipette into 1st well of gel
  10. Mix 2 uL of sample with 1 uL of loading dye pipette 2 uL into the next well
  11. Load all samples into the gel in the same manner
  12. Run gel at 300 V for 14 minutes
PCR
Backbone
  1. Centrifuge primers for 30 seconds at 13,000 rpm
  2. Add 40 uL of 5X Phusion HF Buffer
  3. Add 40 uL of 10 mM dNTPs
  4. Add 5 uL of the forward primer and 5 uL of the reverse primers
  5. Add 2 uL of the template DNA (pAWP78)to the tube
  6. Add 6 uL of DMSO
  7. Add 2 uL of Phusion DNA Polymerase
  8. Mix well
  9. Aliquot 25 uL of reaction mixture into 8 PCR tubes
  10. Set up a PCR gradient from 55 ºC to 75 ºC, with an extension time of 2 minutes and 30 seconds
Colony
  1. Centrifuge primers for 30 seconds at 13,000 rpm
  2. Thaw CloneID Master Mix on ice
  3. Add 0.2 uL of forward and 0.2 uL reverse primer
  4. In PCR tubes, add 20 uL CloneID and primers to each tube
  5. Take a pipette tip, and gently swab a colony from each plate
  6. Swab using the pipette tip and place into the CloneID tubes, pipette up and down to mix and swirl
  7. Add 50 uL of LB to PCR tubes (same number as PCR reactions)
  8. Transfer 1 uL of the PCR+cells (CloneID) solution to the mini cultures with 50 uL LB and put into 37 ºC incubator
  9. Load PCR reactions into the thermocycler

PCR cycle:

Temp (ºC) Time
Lysis x 1 98 2 min
Denaturation x 35
Annealing x 35
Elongation x 35 		98
52
72 		20 sec
15 sec
4 min
Final x 1 72 10 min
Hold 4 indefinite
Gibson Assembly
  1. Calculate the optimal amounts of DNA for assembly: pmols =(weight in ng)x1000/ (base pairs x 650 daltons)
  2. NEB recommends at least a 2-3:1 molar ratio of insert to backbone for 1-2 insert(s); up to a 1:10 ratio can be used. They also recommend between 0.01-0.25 pmol total DNA in the reaction.
  3. Using the ratios and dilution factors calculate the appropriate amount of microliters of backbone and inserts to add
  4. Add x uL of backbone to the PCR tube
  5. Add x uL of the insert to the PCR tube
  6. Add x uL dd H20 to the PCR tube to reach a volume of 5 uL
  7. Add 5 uL of NEB HiFi DNA Assembly Mix to the PCR tube (1:1 of Assembly mix to solution)
  8. Incubate the reaction for 30 minutes for 3 part assembly and 60 minutes for 4 part assembly, at 50 ºC
  9. Place in the -20 ºC freezer

The following tables designate attempt 1 for the anoxic group, further attempt ratios are detailed in the lab notebook:

Gibson 1 (Construct 1) :

Fragment Amount (uL)
pmoA- RBS-nir 1 uL
RBS-DAMO_2434 1 uL
pAWP78 backbone 1.9 uL

Gibson 2 (Construct 2) :

Fragment Amount (uL)
pmoA-RBS-nir 1 uL
RBS-DAMO_2437 1 uL
pAWP78 backbone 1.9 uL

Gibson 3 (Construct 3) :

Fragment Amount (uL)
pmoA-RBS-nir 1 uL
RBS-DAMO_2434 1 uL
RBS-DAMO_2437 1 uL
pAWP78 backbone 1.9 uL
Growth Assay
  1. Move all needed materials into the anaerobic chamber and allow to sit over several days: P media, 18 tubes, stoppers, metal clamps, methanol, and milliQ water
  2. Place 1 mL of dd H2O in an Eppendorf tube
  3. Take a swab of 20Z and mix it into the Eppendorf tube
  4. In the anoxic chamber, add 5 mL of P media to each tube
  5. Add 75 uL of methanol to 9 of the tubes
  6. Pipette 50 uL from the inoculum into each of the tubes
  7. Cap tubes with metal lid and remove from chamber
  8. With a 10 cc syringe, 0.2 micron filter, and needle, remove 10 mL of air from the anoxic methane tube, 20 mL from microaerophilic methane tube and 10 mL from microaerophilic methanol
  9. For tubes with methane, add methane gas for 10 seconds using a needle
  10. Add 10 mL of oxygen to oxic methane tube and microaerophilic methane and methanol tubes
  11. Take initial optical density for all tubes and shake overnight at 200 rpm at 30 ºC
  12. Continue analyzing growth overtime to establish growth rate

When completed there should be 18 tubes total of the following (each group done in triplicate):

  • 3 Oxic Methane
  • 3 Microaerophilic Methane
  • 3 Anoxic Methane
  • 3 Oxic Methanol
  • 3 Microaerophilic Methanol
  • 3 Anoxic Methanol
Bacteria Transformations
via Heat Shock
  1. Spin down the PCR tubes with constructs in the centrifuge
  2. Pipette 4.5 uL from each PCR tube into its respective .5 mL eppendorf tube under sterile conditions
  3. Keep on ice for 20 minutes
  4. Prepare the heat shock system finding a way to maintain 40 ºC
  5. Heat shock for 1 minute at 40 ºC
  6. Put on ice for 2 minutes
  7. Immediately afterwards add 500 uL of LB media to each eppendorf tube under sterile conditions
  8. Transfer the solution to a 1.5 mL eppendorf tube using a micropipette
  9. Shake for 1 hr at 30 C
  10. Spin down the cells at 13,000 rpm for 2 min
  11. Dump excess volume into liquid waste
  12. Resuspend the pellet using a micropipette
  13. Using a micropipette, put the resuspended pellet onto the LB + antibiotic plate
  14. Spread over the entirety of the plate using a disposable spreader
  15. Repeat for each sample
  16. Incubate overnight at 37 ºC
via Electroporation
  1. Add 50 uL of competent E. Coli cells to each cuvette avoiding bubbles
  2. Add 50 uL of dd H20 to an electroporation cuvette under sterile conditions
  3. Add 5 uL of the assembled constructs to the cuvette
  4. Put the cuvette into the electroporator, electroporate each cuvette for a few seconds
  5. Add 1 mL of LB media to each cuvette, resuspending the competent cells
  6. Pipette the solution from the cuvette to a clean Eppendorf tube
  7. Incubate the eppendorf tube at 37 C, shaking, for 1 to 1.5 hrs at 600 rpm
  8. Centrifuge at 13,000 rpm for 1 minute to create a cell pellet and decant excess liquid
  9. Resuspend the cell pellet and pipette each sample onto an LB + antibiotic plate
  10. Use a spreader to finish plating
  11. Incubate the plates at 37 C overnight
Making Competent Cells
Calcium Competent Cells

Day 1:

  1. Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Work sterile. Grow plate overnight at 37°C.

Day 2:

  1. Autoclave:
    1 L LB (or your preferred media)
    • 1 L of 100 mM CaCl2
    • 1 L of 100 mM MgCl2
    • 100 mL of 85 mM CaCl2, 15% glycerol v/v
    • 4 centrifuge bottles and caps
    • Lots of microfuge tubes
  2. Chill overnight at 4°C:
    • 100 mM CaCl2 100 mM MgCl2
    • 85 mM CaCl2, 15% glycerol v/v
    • Centrifuge rotor
  3. Prepare starter culture of cells
    Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media – no antibiotics). Grow culture at 37°C in shaker overnight.

    4. Notes:

    • You will have extra CaCl2 and MgCl2. These solutions can be saved and reautoclaved for the next time you make competent cells.
    • You can also substitute other media like SOB, 2xYT, etc. for the LB if you prefer.
    • All glassware should be detergent free. Presence of detergent reduces competency of cells.

Day 3:

  1. Inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker. Measure the OD600 every hour, then every 15-20 minutes when the OD gets above 0.2.
  2. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.

    3. IMPORTANT NOTES:

    • It is important not to let the OD get any higher than 0.4. The OD should be carefully monitored and checked often, especially when it gets above 0.2, as the cells grow exponentially. It usually takes about 3 hours to reach an OD of 0.35 when using a 10 mL starter culture.
    • It is also very important to keep the cells at 4°C for the remainder of the procedure. The cells, and any bottles or solutions that they come in contact with, must be pre-chilled to 4°C.

  3. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g (~4000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4°C.
  4. Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.
  5. (Spin #2) Harvest the cells by centrifugation at 2000g (~3000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4°C.
  6. Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
  7. (Spin #3) Harvest the cells by centrifugation at 2000g (~3000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4°C. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.
  8. Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85 mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.
  9. (Spin #4) Harvest the cells by centrifugation at 1000g (~2100 rpm in the Beckman GH-3.8 rotor) for 15 minutes at 4°C.
  10. Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85 mM CaCl2, 15% glycerol. The final OD600 of the suspended cells should be ~ 200-250.
  11. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.
Electrocompetent Cells

Requirements:

  • Eppendorf tubes (Ice cold)
  • Refrigerated centrifuge (4°C)
  • Sterile 10% glycerol (Ice cold)
  • The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).

Media Protocol for 250 mL cultures:

  1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of LB or TB or SOB media in a 125 ml flask and incubate for 16-18 hours at 37°C and 250 rpm.
  2. Have ready 1 L flask containing 250 ml each of LB or TB or SOB media pre-warmed to 37°C. Add 1% of the overnight culture to the flask.
  3. Shake at 37°C and 250 rpm until the culture reaches an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 500 mL of 10% glycerol on ice well in advance of harvesting cells.
  4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
  5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
  6. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
  7. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
  8. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
  9. At this point you can electroporate or freeze the cells away. To freeze, add 50 microliters of the culture to microcentrifuge tubes on ice.
  10. Once you have used all of the culture, transfer the tubes to liquid Nitrogen for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months.
Harvesting Cells
  1. Sterile conditions are not needed
  2. Label all eppendorf tubes, one for each test tube
  3. Pipette 1 mL of MPM culture into each test tube
  4. Put all tubes in centrifuge for 3 minutes at 13,300 RPM
  5. Pour all the liquid from tubes into liquid waste disposal
  6. Repeat steps 3-5 until all MPM culture has been centrifuged, about 5 mL
  7. Pipette out all liquid and dispose in liquid waste
  8. Add 500 uL of MilliQ water to each test tube
  9. Centrifuge for 5 minutes
  10. Pipette out all access water, leaving just the pellet at the bottom of the tube
  11. Put all tubes in the 65 degree isotemp oven incubator
DPN1 Digestion
  1. Combine 1 uL of DPN1 digest with 50 uL of your solution; adjust DPN1 based on solution volume
  2. Incubate at 37 ºC for one hour
  3. Dilute volume of reaction buffer to 200 uL with ddH2O
  4. Add 100 uL of binding buffer to the tube. Mix thoroughly by pipetting.
  5. Add 300 uL of pure ethanol to the tubes and mix thoroughly
  6. Transfer solution to DNA purification microcolumn
  7. Centrifuge column for 60 seconds at 13,000 rpm
  8. Discard flow-through
  9. Add 700 uL of wash buffer to column and centrifuge for 60 seconds at 13,000 rpm
  10. Discard flow-through
  11. Repeat wash steps 9-10
  12. Centrifuge empty column for 1 minute
  13. Transfer microcolumn to a clean Eppendorf tube
  14. Add 10 uL of ddH2O
  15. Centrifuge for 1 minute at 13,000 rpm
  16. Store purified DNA at -20 C