Team:Bulgaria/Protocols

Making Electrocompetent Cells +

  • Day 1

    Streak out frozen glycerol stock of E. coli (Top 10, DH5α, etc.) onto an LB plate (no antibiotics). Grow plate overnight at 37°C.

  • Day 2

    Autoclave: 2 L of ddH2O 100 mL of 10% v/v glycerol (molecular biology grade), 1 L LB media, 4 centrifuge bottles and caps plus lots of 1.5 ml tubes

  • Day 3

    1. Autoclave: 2 L of ddH2O 100 mL of 10% v/v glycerol (molecular biology grade), 1 L LB media, 4 centrifuge bottles and caps plus lots of 1.5 ml tubes
    2. Chill overnight at 4°C: ddH2O, 10% glycerol, centrifuge rotor
    3. Prepare starter culture - select a single colony of from fresh LB plate and inoculate a 10 mL starter culture of LB. Grow culture at 37°C in shaker overnight.

  • Day 4

    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. (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 1000g for 20 minutes at 4°C.
    4. Decant the supernatant and resuspend each pellet in 200 mL of ice cold ddH2O.
    5. (Spin #2) Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C.
    6. Decant the supernatant and resuspend each pellet in 100 mL of ice cold ddH2O.
    7. (Spin #3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. At this step, rinse two 50 mL conical tubes with ddH2O and chill on ice.
    8. Decant the supernatant and resuspend each pellet in 40 mL of ice cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
    9. Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
    10. Carefully aspirate the supernatant with a sterile Pasteur pipette. Resuspend each pellet in 1 mL of ice cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be ~ 200-250.
    11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cell in the -80°C freezer.

Electroporation Protocol +

  1. Place SOC recovery medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour. Place electroporation cuvettes (2mm) and microcentrifuge tubes on ice.
  2. As a positive control for transformation, use the 20 pg/μl plasmid from the iGEM kit. Heat-denatured ligation reactions can be used for electroporation directly; however, column purification is recommended.
  3. Use 100 μl freshly prepared electrocompetent cells in a pre-chilled microcentrifuge tube. Add 1 μl of the DNA solution.
  4. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. Electroporate using the Prokaryotes O program for Eppendorf Eporator.
  5. Immediately add 500 µl of 37°C SOC to the cuvette, gently mix up and down twice, then transfer to a 1.5 ml tube.
  6. Shake vigorously (250 rpm) or rotate at 37°C for 1 hour.
  7. Dilute the cells as appropriate then spread 10-200 μl cells onto a pre-warmed selective plate.
  8. Incubate plates overnight at 37°C.

Plasmid DNA isolation - Miniprep +

  1. Apply 40 μl of activation Buffer PL onto the spin-column (do not spin) and keep it at room temperature till transfering lysate to the spin-column.
  2. Pour cells from overnight culture (11-14 h) into 1.5-2 ml tubes. Spin down 1.5-4 ml in a microcentrifuge at app. 14,000 rpm for 2 minutes. Pour off the supernatant and blot tubes upside-down on paper towel to remove any remaining media.
  3. Add 250 µl of Cell R buffer and completely resuspend the cell pellet.
  4. Add 200 µl of blue-coloured Lysis Blue buffer. Mix gently, but completely by several-fold inverting, until uniform blue colour of cell resuspension is obtained.
  5. Add 350 µl of neutralization and binding buffer Neutral B. Mix by several-fold inverting, until blue colour will disappear.
  6. Spin down in a microcentrifuge at app. 14,000 rpm for 7 minutes.
  7. Pour the supernatant from step 5 into the spin-column placed in the receiver tube.
  8. Spin down at 12,000 rpm for 1 minute.
  9. Remove the spin-column, pour off supernatant and place back into the receiver tube.
  10. Add 500 µl of Wash PLX1 buffer and spin down at 12,000 rpm for 1 minute.
  11. Remove spin-column, pour off supernatant, replace back spin-column.
  12. Add 650 µl of Wash PLX2 buffer and spin down at 12,000 rpm for 1 minute.
  13. Remove spin-column, pour off supernatant, replace spin-column.
  14. Spin down at 12,000 rpm for 2 minutes to remove traces of the Wash PLX2 buffer.
  15. Place spin-column into new receiver tube (1.5-2 ml). Add 50-100 µl of Elution buffer to elute bound DNA.
  16. Incubate spin-column/receiver tube assembly for 2 minutes at room temperature.
  17. Spin down at 12,000 rpm for 2 minutes.
  18. Remove spin-column, cap the receiver tube. Plasmid DNA is ready for analysis/manipulations.
  19. Remove spin-column, cap the receiver tube. Plasmid DNA is ready for analysis/manipulations. It can be stored either at 2÷8oC or (preferred) at -20oC

gBlocks Fragment Amplification +

  1. Phusion® DNA Polymerase amplification reaction

    Nuclease-free H2O Adjust to final 50 μL 5X Phusion HF 10 μL 10 mM dNTPs 1 μL 10 μM Forward Primer 2.5 μL 10 μM Reverse Primer 2.5 μL gBlocks® Gene Fragments 0.1–1.0 ng Phusion® DNA Polymerase 0.5 μL Total volume 50 μL

  2. PCR program:

    Initial denaturation: 98˚C, 30 seconds Cycle start: Denaturation 98˚C, 10 seconds Annealing 45–72˚C, 10–30 seconds Extension 72˚C, 20 seconds per kb Cycle end (20 cycles) Final extension 72˚C, 5 minutes Hold 4˚C, ∞

PCR Purification of DNA fragment (Quick Gel Extraction and PCR Purification Combo Kit) +

  1. Add 4 volumes of Binding Buffer (B2) to 1 volume of PCR reaction (50–100 μL). Mix well.
  2. Remove a PureLink® Clean-up Spin Column in a Wash Tube from the package.
  3. Add sample in Binding Buffer from step 1, above, to the PureLink® Spin Column.
  4. Centrifuge the PureLink® Spin Column at room temperature at 10,000 × g for 1 minute.
  5. Discard the flow through and replace the PureLink® Spin Column into the Wash Tube.
  6. Add 650 μL Wash Buffer with ethanol to the PureLink® Spin Column.
  7. Centrifuge the PureLink® Spin Column at room temperature at 10,000 × g for 1 minute. Discard the flow-through from the Wash Tube and replace the PureLink® Spin Column into the tube.
  8. Centrifuge the PureLink® Spin Column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the Wash Tube.
  9. Place the PureLink® Spin Column in a clean 1.7-mL PureLink® Elution Tube (supplied with the kit).
  10. Add 50 μL Elution Buffer (E1) or sterile, distilled water (pH >7.0) to the center of the PureLink® Spin Column.
  11. Incubate the PureLink® Spin Column at room temperature for 1 minute.
  12. Centrifuge the PureLink® Spin Column at maximum speed for 1 minute.
  13. The elution tube contains the purified PCR product. Remove and discard the PureLink® Spin Column. The recovered elution volume is ~48 μL.

Purifying DNA from Gels Using a Centrifuge +

  1. Pipet the dissolved gel piece containing the DNA fragment of interest (steps 4–5, page 10) into the center of a PureLink® Clean-up Spin Column inside a Wash Tube. Note: Do not load >400 mg agarose per PureLink® Spin Column.
  2. Centrifuge the tube at >10,000 × g for 1 minute. Discard the flow-through and replace the PureLink® Spin Column into the Wash Tube.
  3. Add 500–700 μL Wash Buffer (W1), containing ethanol, to the PureLink® Spin Column.
  4. Centrifuge the tube at >10,000 × g for 1 minute. Discard the flow-through and replace the PureLink® Spin Column into the Wash Tube.
  5. Centrifuge the tube again at maximum speed for 2–3 minutes to remove any residual Wash Buffer and ethanol.
  6. Discard the Wash Tube and place the PureLink® Spin Column into an Elution Tube.
  7. Add 50 μL Elution Buffer (E1) to the center of the PureLink® Spin Column.
  8. Incubate the tube for 1 minute at room temperature.
  9. Centrifuge the tube at >10,000 × g for 1 minute. The Elution Tube contains the purified DNA. Discard the PureLink® Spin Column.
  10. Store the purified DNA or proceed to your downstream application of choice.

Overlap extension PCR +

  1. Set up PCRs using Phusion High Fidelity Polymerase to generate products AB and CD.
  2. Gel purification of products X and Y
  3. Component Product XY amplification reaction Nuclease-free H2O Adjust to final: 50 μL 5X Phusion HF : 10 μL 10 mM dNTPs: 1 μL Product X (template): 50–125 ng Product Y (template): 50–125 ng 10 μM Forward Primer (X forward): 2.5 μL 10 μM Reverse Primer (Y reverse): 2.5 μL Phusion polymerase: 0.5 μL

Protocol for Restriction Digestion +

Add: 2 µL 10X Buffer (according to the enzymes specifications). X µL DNA (0.5-1 µg/µL) 0.5-2 µL selected restriction enzyme nuclease-free water up to 20 µL Mix gently and spin down for a few seconds. Incubate at 37°C for 1-16 hours (according to the enzymes specifications and the DNA amounts). The digestion reaction may be scaled either up or down.

Ligations +

  1. Add the following reagents to a PCR tube: Reagent Amount 5X Ligase Reaction Buffer Vector DNA 50-100 ng Insert DNA (1:1 or 3:1 ratio) ExpressLink™ T4 DNA Ligase 5 units (in 1 μL) autoclaved distilled water to 20 μL
  2. Mix gently. Centrifuge briefly to bring the contents to the bottom of the tube.
  3. Incubate at room temperature for 5 - 15 minutes.
  4. Use 2 μL of the ligation reaction to transform 100 μL of electrocompetent cells.

Glycerol Stocks +

  1. Pick a single colony of the clone off of a plate and grow an overnight in the appropriate selectable liquid medium (e.g., LB amp).
  2. Make a label (clone ID # and date) for the construct.
  3. Add 0.5ml of the o/n culture to 0.5ml of 80% sterile glycerol in the sterile screw cap microcentrifuge tube.
  4. Screw a lid onto the tube and write the clone ID # on the lid of the tube.
  5. Vortex.
  6. Freeze the glycerol stock at –80oC.

  7. To streak out from a glycerol stock:

    1. Determine the location (-80oC tower #, box #, row #) of the construct.
    2. Take the tube to the place that you intend to streak the clone out
    3. Flame a metal inoculating loop until it is red hot.
    4. Scrape off a portion from the top of the frozen glycerol stock and streak it onto your plate.
    5. Return the construct to the –80oC.

Induction and Purification of proteins: +

We used Rosetta-gami (DE3) pLysS cells. The pLysS plasmid, carried by those cells, has a chloramphenicol resistance so we added chloramphenicol to the growth media. In case of other expression strains (e.g. BL21) without chloramphenicol resistance this antibiotic should not be added.

  1. Take a single colony and inoculate 2ml LB / Amp + Chloramphenicol at 37oC over night
  2. Next morning dilute 1:50 or 1:100 in a 5ml LB / Amp + Chloramphenicol and incubate at 37oC with shaking at 230rpm.
  3. Wait till the OD gets to 0.4-0.5 and take out 0.5ml of culture, freeze pellet- store. This is the uninduced time point.
  4. Add this point add 1mM (final concentration) of IPTG.
  5. After adding 1mM IPTG, split the culture - take out 1ml and incubate that tube at 20oC with shaking at 200rpm for 16 hours. Re-incubate remaining culture at 37oC with shaking at 230 rpm, and collect 0.5ml fractions every hour for 3 hours.
  6. Spin down the cultures (including the un-induced) at max speed and resuspend The cell pellet in 50ul of SDS sample buffer.
  7. Boil the samples for 3 minutes with brief vortexing every minute.
  8. Load 5 ul (equivalent to 100ul of cells) onto an SDS –PAGE gel, and Coomaasie stain the gel.

SDS-PAGE