Team:UC San Diego/Protocols

Background

Gibson Assembly (NEB)

  1. Set up the reactions on ice based on the calculator.
  2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
  3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

Transformation (NEB)

  1. Thaw tube of DH5-alpha cells on ice for 10 minutes.
  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 45 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pipette 120 µl of room temperature LB broth into the mixture.
  7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C.
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours

PCR (Novagen)


  1. In PCR tubes combine the following:

    2. Component Volume Final Concentration
    10X Buffer for KOD Hot Start DNA Polymerase 5 ul 1X
    25 mM MgSO4 3 ul 1.5 mM
    dNTPs(2mM each) 5 ul 0.2 mM
    PCR Grade Water X ul
    Sense (5') Primer (10 uM) 1.5 ul 0.3 uM
    Anti-Sense (3') Primer (10 uM) 1.5 uL 0.3 uM
    Template DNA Y ul
    KOD Hot Start DNA Polymerase (1 U/ul) 1 ul 0.021 U/ul
    Total Reaction Volume 50 ul

  2. Transfer PCR tubes to a thermocycler and begin thermocycling:

    3. Step Target Size
    <500 bp 500-1000bp 1500-3000bp >3000bp
    1. Polymerase Activation 95 C for 2 min 95 C for 2 min 95 C for 2 min 95 C for 2 min
    2. Denature 95 C for 20 s 95 C for 20 s 95 C for 20 s 95 C for 20 s
    3. Annealing Lowest Primer Tm C for 10s
    4. Extension 70 C for 10s/kb 70 C for 15s/kb 70 C for 20s/kb 70 C for 25s/kb
    Repeat Steps 2-4 20-40 cycles

    For site-directed mutagenesis, use non-overlapping, phosphorlyated primers containing the desired mutation. Ligate the PCR product and transform.

PCR Purification (Thermofisher)

  1. Add 4 volumes of PureLink® Binding Buffer (B2) with isopropanol or Binding Buffer HC (B3) with isopropanol to 1 volume of the PCR product (50–100 μL). Mix well.
  2. Remove a PureLink® Spin Column in a Collection Tube from the package.
  3. Add the sample with the appropriate Binding Buffer (from step 1 of this procedure) to the PureLink® Spin Column.
  4. Centrifuge the column at room temperature at 10,000 × g for 1 minute.
  5. Discard the flow through and place the spin column into the collection tube.
  6. Add 650 μL of Wash Buffer with ethanol to the column.
  7. Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
  8. Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube.
  9. Place the spin column in a clean 1.7-mL PureLink® Elution Tube supplied with the kit.
  10. Add 50 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile, distilled water (pH >7.0) to the center of the column.
  11. Incubate the column at room temperature for 1 minute.
  12. Centrifuge the column at maximum speed for 2 minutes.
  13. The elution tube contains the purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μL.
  14. Store the purified PCR product at –20°C or use the PCR product for the desired downstream application.

Miniprep (QIAGEN)

  1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source.
  7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
  8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source. Transfer the QIAprep spin column to the collection tube.
  9. Centrifuge for 1 min to remove residual wash buffer.
  10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Gel Electrophoresis (Addgene)

    Pouring a Standard 1% Agarose Gel:
  1. Measure out 1g of agarose.
  2. Pour agarose powder into microwavable flask along with 100mL of 1xTAE.
  3. Microwave for 1-3min (until the agarose is completely dissolved and there is a nice rolling boil).
  4. Let agarose solution cool down for 5min.
  5. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5μg/mL (usually about 2-3μl of lab stock solution per 100mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
  6. Pour the agarose into a gel tray with the well comb in place.
  7. Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 minutes, until it has completely solidified.
    Loading Samples and Running an Agarose Gel:
  1. Add loading buffer to each of your digest samples.
  2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
  3. Fill gel box with 1xTAE (or TBE) until the gel is covered.
  4. Carefully load a molecular weight ladder into the first lane of the gel.
  5. Carefully load your samples into the additional wells of the gel.
  6. Run the gel at 80-150V until the dye line is approximately 75-80% of the way down the gel.
  7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
  8. Using any device that has UV light, visualize your DNA fragments.

Gel Purification (Zymo Research)

  1. Excise the DNA fragment1 from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube.
  2. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB).
  3. Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved. For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 µl agarose, 300 µl ADB, and 100 µl water).
  4. Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.
  5. Centrifuge for 30-60 seconds. Discard the flow-through.
  6. Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. Repeat the wash step.
  7. Add ≥ 6 µl DNA Elution Buffer or water directly to the column matrix. Place column into a 1.5 ml tube and centrifuge for 30-60 seconds to elute DNA. Ultra-pure DNA is now ready for use.

Restriction Digest (Addgene)

  1. In a 1.5mL tube combine the following:
    DNA
    Restriction Enzyme(s)
    Buffer
    dH2O up to total volume
  2. Mix gently by pipetting.
  3. Incubate tube at appropriate temperature (usually 37°C) for 1 hour. Always follow the manufacturer’s instructions.

Ligation (NEB)

  1. Combine 50 ng of vector with a 3-fold molar excess of insert. Use NEBioCalculator to calculate molar ratios. Adjust volume to 10 μl with dH2O.
  2. Add 10 μl of 2X Quick Ligation Buffer and mix.
  3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
  4. Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
  5. Chill on ice, then transform or store at -20°C.
  6. Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.