Team:CCA SanDiego/procedures.html

Procedures - CCA_SANDIEGO iGEM

AGAR PLATE PREPERATION
  1. Weigh out agar.
  2. Pour agar into glass bottle.
  3. Measure out DI water.
  4. Pour out water into glass bottle while washing out agar weigh boat.
  5. Stir, put in microwave.
  6. Microwave on high, every 1 min take out and stir. Microwave until agar is clear and bubbling.
  7. Lay out Petri dishes.
  8. Label on bottom.
  9. Use pipette 35 mL to aliquot LB solution to plates. ​
  10. Put on lids, let sit for 30 min.
  11. Store upside down at room temperature or 4 degrees Celsius.
RESTRICTION DIGEST:
Setting up 50 microL reaction:
  • 1 microL RE 1
  • 1 microL RE 2
  • 5 microL CutSmart Buffer
  • ​1 microgram DNA
Pipette up and down, shake a little bit.

Incubate for 1 hr in 37 degrees Celsius incubator. Heat Kill at 65 degrees C (or 80 degrees Celsius if HIndIII is used) for 20 min.
LIQUID CULTURE
  1. Get 10 mL tube.
  2. Add 5 mL of LB into 10 mL tube. Add 5 microL of 35 mg/mL Chl drug stock to LB. Swirl and shake.
  3. Use sterile inoculating loop to scrape one colony off of selective plasmid plate.
  4. Submerge the end of the loop with the colony into the LB and swirl.
  5. Place into shaker with shaking and heat on overnight.
GEL ELECTROPHORESIS
  1. Prep 1% gel.
  2. Orient gel correctly.
  3. Add TBE buffer.
  4. Add 3 microL EtBt to buffer solution and swirl.
  5. Add 10 microliter dye to 50 microliter DNA solution.
  6. Load DNA into wells.
  7. Run gel at 100 V for 1.5 hours.
  8. Pick up and transfer for imaging, cut out appropriate band.
LAST WEEK OF EXPERIMENTS:
DAY 1:
  1. Resuspend well 13J in Kit Plate 1 and well 12P in Kit Plate 6 with 10 µl of Ultrapure water, transform one 50 aliquot of bacteria using heat shock protocol.
  2. Restriction digest for the second miniprepped NPR sample using the enzymes XbaI and HindIII
    1. Run out on gel when finished and cut out smaller fragment.
    2. Store gel fragment in fridge.
  3. Prep two more gels.
  4. Make stock drug solution of 35 mg/mL using ethanol.
DAY 2:
  1. Check colonies. Choose 1 colony from 1 plate, do liquid culture with appropriate amount of chloramphenicol added to the liquid culture. Use amp for the CYP2e1 for liquid culture
    1. 5 mL LB per tube.
    2. Put in shaker overnight.
  2. Liquid culture for CYP2e1 bicistronic.
  3. Gel purification from sample from day 1.
DAY 3:
  1. Miniprep for 13J/12P and CYP2e1
    1. Save CYP2e1 in fridge.
  2. Restriction digest miniprep product (13J/12P only) with XbaI and SpeI.
    1. Run gel.
  3. Ligate digested 13J/12P with HindIII bridge (1000 ng/microL) and CPR fragment.
    1. Dilute bridge with 1xTE buffer to 100 pg/microL.
    2. 20 microliter ligation.
    3. Save 10 microL of sample to dry down overnight in the hood.
  4. Transform 50 µl aliquot of bacteria with 5 µl ligation product. Transform 50 µl aliquot of bacteria with CYP2e1 bicistronic.
DAY 4:
  1. Check colonies under UV and visible light for non colorful (white) colonies on the ligation plate. Select that colony and do liquid culture.
DAY 5:
  1. Miniprep from liquid culture for CYP2e1/CPR and ligation product.
  2. Dry ligation product.

TRANSFORMATION 
  1. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  2. Add 1-5 µl containing 1 pg - 100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.

  5. Pipette 950 µl of room temperature SOC into the mixture.
  6. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  7. Warm selection plates to 37 degrees Celcius.
  8. Mix the cells thoroughly by flicking to the tube and inverting.
  9. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
PLASMID MINIPREP:
  1. Centrifuging 2 mL of overnight culture at 12000 speed for around 1 min.
  2. Remove supernatant. Resuspend with 250 µl of R3 to cell pellet until mixture is homogenous.
  3. Add 250 µl L7 to step 2. Mix by inverting gently. Incubate at room temp for 5 min.
  4. Add 350 µl N4 and mix immediately until homogenous. Centrifuge at 12000 speed for 10 min.
  5. Take supernatant from step 4 and transfer to spin column in wash tube. Centrifuge at 12000 speed for 1 min. Discard wash tube and place column in another wash tube.
  6. Add 500µl W10 into column. Incubate column for 1 min at room temperature. Centrifuge column at 12000 speed for 1 min. Discard wash tube and place column in another wash tube.
  7. Add 700 µl W9 into column. Centrifuge at 12000 speed for 1 min. Discard the flowthrough and place column back in the wash tube. Centrifuge for 1 min at same speed. Discard wash tube with the flowthrough.
  8. Place spin column in 1.5 mL tube. Add 75 µl TE buffer to center of column. Incubate for 1 min at room temperature.
  9. Centrifuge column and 1.5 mL tube at 12000 speed for 2 min. Discard column. 1.5 mL tube contains plasmid DNA.