Team:Alberta/Protocols

From 2012.igem.org

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<li>Add 1 mL of LB broth to microcentrifuge tube, and incubate for 1 hour at 37°C
<li>Add 1 mL of LB broth to microcentrifuge tube, and incubate for 1 hour at 37°C
<li>Plate 200 µL of culture on selective plates, such as chloramphenicol, kanamycin, or any combination with appropriate concentrations (see LB Agar Plates for our concentration)   
<li>Plate 200 µL of culture on selective plates, such as chloramphenicol, kanamycin, or any combination with appropriate concentrations (see LB Agar Plates for our concentration)   
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Digestion and Ligation
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<li>Create plasmid DNA with concentration between 20 and 200 ng/µL for optimum digestion
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<li>Transfer 200 ± 10 ng of plasmid DNA to microcentrifuge tube
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<li>Add 1 µL enzyme for single digest, and an additional 1 µL different enzyme for double digest
 +
<li>Add 2 µL of 10X appropriate buffer
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<li>Fill microcentrifuge tube with milliQ water to reach 20 µL final volume
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<li>Incubate for 1 hour at 37°C, or longer if appropriate buffer cannot be used
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<li>Refer PCR/Digestion/Ligation Clean-up for purifying digested DNA in preparation of  ligation
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<li>Add 3 µL T4 DNA Ligase 10X buffer to 30 µL eluted DNA, then add 1 µL T4 DNA Ligase
 +
<li>Keep mixture at room temperature for 1 hour
 +
<li>If transforming, use 10 µL of ligated DNA for transformation
 +
<li>Refer PCR/Digestion/Ligation Clean-up if further experiments will be performed
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Revision as of 20:54, 27 August 2012

   Team Alberta




Protocols

Gradient plates

The following procedure is to be used to find values related to diffusion. All time values should be converted to seconds, from when the antibiotic was plated, and all distance values should be recorded in centimeters, from the edge of the well to the first sign of life at the edge of the “kill zone”. All materials should be kept as sterile as possible.

  1. Draw a central cross on the lid of a Petri dish
  2. Sterilize a tall cylindrical magnet of a height near, but not at the depth of the plate (a well that reaches the bottom of the plate will allow the antibiotic in question to seep under the agar rather than diffuse through it) and radius 0.25 cm. Place the sterilized magnet on the inside of the Petri dish lid and, from outside the lid, adjust and secure the sterilized magnet with another magnet.
  3. Melt and pipette 25 mL of LB agar into the Petri dish, and place the lid on top, allowing the magnet to rest in the molten agar. Let agar rest until solid and cool, then remove the cap and allow the surface to dry until it is free of excess moisture.
    Steps 4 and 5 may be completed in any desired order, depending on the approximate amount of time the substance is meant to diffuse for.
  4. Pipette 50 µL of antibiotic (at a concentration high above the minimum inhibitory concentration) into the centre well. Be careful not to spill. Immediately place the plate in a 37ºC incubator.
  5. Evenly plate 200 µL of cells with a resistance to the antibiotic over the flat surface of the plate. Immediately place the plate in a 37ºC incubator.
  6. Begin watching for results within 2 hours of plating. These will come in the form of a slight difference in texture between the zone in which cells are growing, and the zone in which they are not. It will be very subtle, and may need to be observed by shining light through the agar, or placing the plate on a black backdrop. As soon as it is observed, the radius of the zone must be measured from the edge of the well. The zone may expand. Continue recording the size and time until it stops changing.

Chemically-Induced Competence

The protocols described below were used to create competent cells of Top10 and TG-1 Escherichia coli strains. The Calcium Chloride protocol uses less steps, is easier to perform, and produces competent cells faster than the Liquid Nitrogen procedure. However, we found that the competence efficiency was higher using the Liquid Nitrogen protocol.


Calcium chloride

  1. Introduce cells to culture tube containing 5 mL LB medium
  2. Shake overnight at 37°C
  3. Slate 200 µL of culture on separate LB plates
  4. Incubate overnight at 37°C
  5. Add 1.5 mL of 50 µM CaCl2 into microcentrifuge tube
  6. Cool tube on ice for a minimum of 10 minutes
  7. Scrape colonies off plate until 1x0.5 cm smear is achieved
  8. Swirl scraper in CaCl2 until all cells removed, then vortex tube


Liquid Nitrogen

  1. Introduce cells to two culture tubes, each with 5 mL LB medium (one culture tube may be used if large enough for aeration)
  2. Shake overnight at 37°C
  3. Add both cultures to a flask containing 250 mL LB medium
  4. Shake at 37°C for 3-4 hours, checking periodically until 0.55 ± 0.05 at OD600 is reached
  5. Place flask on ice for 10 minutes, then transfer to centrifuge bottles
  6. Spin at 2500x g for 10 minutes at 4°C
  7. Decant supernatant, then add 80 mL ice-cold Inoue transformation buffer
  8. Resuspend cells by swirling slowly, not vortexing
  9. Spin at 2500x g for 10 minutes at 4°C
  10. While spinning, chill one hundred microcentrifuge tubes at -80°C
  11. Decant supernatant, resuspend in 20 mL ice-cold Inoue transformation buffer
  12. Mix DMSO (7% of final volume in bottle), then store on ice for 10 minutes
  13. Transfer 200 µL to each pre-chilled tube
  14. Dip tubes in liquid nitrogen for 5 seconds, then store at -80°C


Plasmid Purification

The following protocol is taken from the instructions provided by Qiagen’s QIAprep Spin Miniprep Kit. We changed the rpm of centrifuge from 13,000 to 14,000, and used a vacuum apparatus for select steps, instead of centrifuge.


  1. Prepare overnight cultures of E. coli in 5 mL LB medium (optional: chemical selection)
  2. Transfer culture to microcentrifuge tube
  3. Spin for 1 minute at 14,000 rpm at room temperature, then decant supernatant
  4. Transfer culture, spin, and decant again, if needed
  5. Resuspend bacterial pellet in 250 µL buffer P1 (which contains RNase A)
  6. Add 250 µL P2 buffer and invert 4-6 times, not vortex
  7. Add 350 µL N3 buffer and invert 4-6 times immediately
  8. Centrifuge for 10 minutes
  9. Pipette supernatant into spin column attached to a vacuum apparatus
  10. Turn on vacuum
  11. Wash spin column with 500 µL of PB buffer
  12. After PB buffer is removed, wash spin column with 750 µL of PE buffer
  13. Remove spin column from vacuum after buffer is fully removed, and attach it to column bottom
  14. Centrifuge for 1 minute to remove residual buffer
  15. Discard column bottom, and attach spin column to 1.5 mL microcentrifuge tube
  16. Add 50 µL of EB/TE or any low salt buffer to center of spin column to elute DNA
  17. After resting for 1 minute, centrifuge for 1 minute
  18. Use a spectrophotometer to measure purity and concentration for storage and for further experiments

Overnight Cultures

  1. Add 5 mL of sterile LB medium to autoclaved 20 mL culture tube
  2. Mix following antibiotic concentration, if needed
    • kanamycin= 1 µL/mL
    • chloramphenicol= 0.6 µL/mL
    • ampicillin= 1 µL/mL
    • tetracycline= 1 µL/mL
  3. Carefully, pick a colony with a sterile pipette tip and eject tip into culture tube
  4. Incubate in shaker at 37°C for a minimum of 12 hours

LB Agar Plates

  1. Add 25 g LB powder mixture to 1 L flask, and fill with 800 mL de-ionized water
  2. Add stir bar to flask and mix on stir plate
  3. Ensure that stir bar is centered in flask, and maintain watch, as flask may fall
  4. After mixture is fully dissolved in water (~10 minutes), add de-ionized water until final volume is 1 L
  5. Add 3.75 g agar into four 500 mL autoclave-safe bottles, and pour 250 mL of the LB mixture into each bottle
  6. Autoclave bottles, and ensure that bottle caps are screwed just loose enough to lift the bottle by grabbing the cap
  7. This will prevent the bottles from exploding from internal pressure, and will also prevent the formation of a vacuum, which can cause contamination
  8. After autoclaving, leave the caps loose until the agar is comfortable to touch
  9. Antibiotics and other chemicals may be mixed in at this stage
    • kanamycin= 1 µL/mL
    • chloramphenicol= 0.6 µL/mL
    • ampicillin= 1 µL/mL
    • tetracycline= 1 µL/mL
  10. Pour 25 mL of molten agar into each sterile petri dish
  11. Let petri dishes cool down until agar solidifies, then it can be used immediately, or stored in fridge
  12. If stored, let agar warm to room temperature before plating, so bacteria are not shocked from rapid temperature changes, leading to less colonies

PCR (50 µl reaction)

  1. Create a working stock (5 ng/µL) of template DNA, and add 1 µl to PCR tube
  2. Add 2.5 µl each of 10 µM forward and reverse primers to PCR tube
  3. Add 1 µl of 25 µM dNTPs
  4. Add 10 µl of 5X HF Phusion buffer, along with 0.5 µL Phusion enzyme
  5. Adjust final tube volume to 50 µl with highly purified water
  6. Try to minimize bubble formation, as this can hinder Phusion from functioning optimally
  7. Set the PCR machine with appropriate denature, annealing, and extension temperatures, as well as appropriate durations and cycles


Gel Electrophoresis

  1. Create a 1 L 1X TAE buffer
  2. Mix 100 mL of 1X TAE buffer with 1 g of agarose to create gel mixture
  3. Microwave for 45 secs, or until solution turns transparent and no agarose is visible
  4. Let solution cool down until comfortable to touch, and pour exactly 16 mL onto a 7.7x6.5 cm glass plate
  5. Ensure that gel mixture spreads evenly and covers entire plate without touching the ground
  6. Place a comb 1.5 cm from the edge of glass, then rest for 10 minutes
  7. Squirt few drops of milliQ water along the interface of comb and gel, then gently remove comb
  8. MilliQ water will prevent vacuum from distorting the lanes, and allows easy removal of comb
  9. Place solidified gel plate in gel apparatus, and fill with enough 1X TAE to fully submerge gel
  10. Create loading solutions of DNA and loading dye, then insert into gel lanes alongside the DNA ladder
  11. Run gel using 150 V, then turn off machine when DNA bands reach 2 cm from end (~20 minutes)
  12. While running, make an Ethidium bromide solution with 2.5 µL Ethidium bromide and 50 mL 1X TAE
  13. Remove the gel from the plate, and transfer only gel to Ethidium bromide solution
  14. After soaking gel for 10 minutes, view banding patterns using UV machine

Transformation of Top10 and TG-1

Refer to Chemically-induced Competence for making competent cells.

  1. Remove competent cells from -80°C freezer, and immediately thaw on ice
  2. Wait until cells fully thaw (~10 minutes) before adding DNA; do not add more than 10% of cell volume
  3. Rest on ice for 30 minutes
  4. Put into 42°C water bath for exactly 90 seconds, then immediately transfer to ice for 2 minutes
  5. Add 1 mL of LB broth to microcentrifuge tube, and incubate for 1 hour at 37°C
  6. Plate 200 µL of culture on selective plates, such as chloramphenicol, kanamycin, or any combination with appropriate concentrations (see LB Agar Plates for our concentration)

Digestion and Ligation

  1. Create plasmid DNA with concentration between 20 and 200 ng/µL for optimum digestion
  2. Transfer 200 ± 10 ng of plasmid DNA to microcentrifuge tube
  3. Add 1 µL enzyme for single digest, and an additional 1 µL different enzyme for double digest
  4. Add 2 µL of 10X appropriate buffer
  5. Fill microcentrifuge tube with milliQ water to reach 20 µL final volume
  6. Incubate for 1 hour at 37°C, or longer if appropriate buffer cannot be used
  7. Refer PCR/Digestion/Ligation Clean-up for purifying digested DNA in preparation of ligation
  8. Add 3 µL T4 DNA Ligase 10X buffer to 30 µL eluted DNA, then add 1 µL T4 DNA Ligase
  9. Keep mixture at room temperature for 1 hour
  10. If transforming, use 10 µL of ligated DNA for transformation
  11. Refer PCR/Digestion/Ligation Clean-up if further experiments will be performed

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