Team:Tec-Monterrey/Experiments

EXPERIMENTS

PCR

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.

Materials

  • Q5 High-Fidelity 2X Master Mix
  • 10 µM Forward Primer
  • 10 µM Reverse Primer
  • DNA Template
  • H2O (Nuclease-free)

Procedure

  • Add Q5 High-Fidelity 2X Master Mix to a PCR tube.
  • Add each Primer to a final concentration of 500 nM.
  • Add up to 10 ng of Template DNA.
  • Add Nuclease-Free Water up to final reaction volume (2 times the volume of master mix).
  • Gently mix the reaction.
    • Alternatively if doing several reactions and using the same amount of template for each one, a pre-mixture of the remaining reagents can be prepared and then distribute the amount of mix in separate tubes prior to adding the template DNA.
  • Collect all liquid to the bottom of the tube by a quick spin if necessary
  • Quickly transfer PCR tubes to a thermocycler previously set to the following configuration:
      Step Temperature Time
      Initial denaturation 98°C 30 Seg
      98°C 10 Seg
      25 to 35 Cycles* 60°C 20 Seg
      72°C 25 Seg
      Final extension 72°C 2 Minutes
      Hold 4°C ∞

      *Use less than 20 cycles if amplifying pure synthesized parts

PCR product purification

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 µL is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g (~13K RPM in a typical microcentrifuge) in a standard laboratory microcentrifuge at room temperature.

Materials

  • Monarch® DNA Cleanup Columns (5 μg)
  • Monarch Collection Tubes (2mL)
  • Monarch® DNA Wash Buffer
  • Monarch® DNA Cleanup Binding Buffer
  • Monarch® DNA Elution Buffer or Nuclease-Free Water
  • Ethanol (≥ 95%)

Before You Begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

Procedure

  • Dilute sample with DNA Cleanup Binding Buffer according to the table:
      Sample type Binding buffer:Sample ratio Example
      dsDNA > 2 kb (plasmids, gDNA) 2:1 200 µL Buffer: 100 µL sample
      dsDNA < 2 kb (some amplicons, fragments) 5:1 500 µL Buffer:100 µL sample
      ssDNA > 200 nt 7:1 700 µL Buffer: 100 µL sample
  • Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100µL is recommended.
  • Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.
  • Re-insert column into collection tube. Add 200µL DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  • Repeat previous step.
  • Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
  • Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
    • Note: Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated

DNA Electrophoresis

QUICKSILVER™ TAE Buffer Powder and SmartGlow PS (E4500) are used for this protocol. Protocol will change with other products.

Materials

  • QUICKSILVER™ TAE Buffer Powder
  • Distilled water
  • SmartGlow PS
  • Agarose
  • SmartCheck Ladder

Procedure

    TAE Buffer (To make 1L of buffer)
  • Add contents of buffer pouch to a graduated cylinder or beaker.
  • Slowly add distilled water to 1 liter.
  • Mix well until all granules are dissolved.
  • Add additional water to bring the volume to 1L, if necessary.
    Agarose Gel (1%)
  • Add 0.5g of agarose to 50 mL of TAE buffer and microwave to dissolve.
  • Add 2.5 µL of SmartGlow and mix.
  • Pour gel into gel tray.
  • Let it sit until solidified and place into the tank.
  • Add TAE buffer into the tank until the gel is covered.
    • NOTE: For better results, 5uL of SmartGlow can be added per 100mL of TAE running buffer.
    Sample preparation and gel observation
  • Add 5-10 µL of Ladder
  • Mix X6 loading dye with samples and DNA markers at a 1:5 (dye to sample) dilution.
  • Run at 100V for 45 to 60min.
  • View the gel using a UV or blue light.

DNA from gel purification

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM). Note that the columns hold up to 800uL.

Materials

  • Monarch® DNA Cleanup Columns (5 μg)
  • Monarch Collection Tubes (2mL)
  • Monarch® DNA Wash Buffer
  • Monarch® Gel Dissolving Buffer
  • Monarch® DNA Elution Buffer or Nuclease-Free Water
  • Ethanol (≥ 95%)

Before You Begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

Procedure

  • Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 mL microcentrifuge tube and weigh the gel slice.
  • Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 µL buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present.
  • Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes).
  • Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
  • Re-insert column into collection tube. Add 200 µL DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  • Repeat previous step.
  • Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
  • Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
    • Note: Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated

Digestion

Procedure

  • Set up reaction as follows:
      Component 20 µL REACTION
      DNA >1 µg
      10X rCutSmart Buffer 2 µL (1X)
      EcoRI-HF 0.5 µL (10 units)
      Nuclease-free Water to 20 µL
  • Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours).
    • For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
    • Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
  • Optional: Heat inactivate at 65°C for 20 min.
    • Heat inactivation is recommended if you plan to continue to another step in the workflow without a DNA purification step.

Double Digestion

Procedure

  • Set up reaction as follows:
      Component 20 µL REACTION
      DNA >1 µg
      10X NEBuffer 2.1 2 µL (1X)
      EcoRI-HF 0.5 µL (10 units)
      PstI 0.75 µL (15 units)
      Nuclease-free Water to 20 µL

      Note: PstI has only 75% of activity in NEBuffer 2.1 and EcoRI has 100%, PstI is added in a ratio of 1.5:1 to EcoRI in order to compensate for the difference.

  • Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours and PstI for at least 2-4 hours).
    • For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
    • Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
  • Optional: Heat inactivate at 80°C for 20 min.
    • Heat inactivation is recommended if you plan to continue to another step in the workflow without a DNA purification step.

Cell-Free protein synthesis

Using a positive control template to verify protein synthesis can be useful when unfamiliar with in vitro transcription-translation protocols. To prevent nuclease contamination, wear gloves and use nuclease-free tubes and tips. Keep all reagents on ice before and during the assembly of reactions and avoid repeated freeze-thaw cycles of the tubes. Reactions are typically 50 μL but can be scaled down or up, as needed. Reactions are typically assembled in nuclease-free 1.5 mL microcentrifuge tubes. Components can be pre-assembled to create a master mix for a desired number of reactions. Use the master mix immediately, discard any unused master mix.

Procedure

  • Thaw all components on ice.
  • Gently vortex the NEBExpress® S30 Synthesis Extract and Protein Synthesis Buffer to mix.
  • Combine reagents in a 1.5 mL microcentrifuge tube on ice as follows:
      Component 50 µL REACTION
      NEBExpress® S30 Synthesis Extract 12 µL
      Protein Synthesis Buffer (2X) 25 µL
      T7 RNA Polymerase 1 µL
      RNase Inhibitor, Murine 1 µL
      Plasmid template (>100 ng/μl) 250 ng
      Nuclease-free Water to 50 µL
  • Incubate reactions at 37°C, with vigorous shaking (250 RPM), for 2-4 hours.
  • Analyze by method of choice or freeze at -20°C for later use.

Competent cell preparation

Materials

  • SOB
    • 0.5% (w/v) yeast extract
    • 2% (w/v) tryptone
    • 10 mM NaCl
    • 2.5 mM KCl
    • 20 mM MgSO4
  • CCMB80 buffer
    • 10 mM KOAc pH 7.0
    • 80 mM CaCl2
    • 20 mM MnCl2
    • 10 mM MgCl2
    • 10% glycerol
    • Adjust pH DOWN to 6.4 with 0.1N HCl if necessary
      • Slight dark precipitate does not affect its function, but makes the next step more difficult.
    • Sterile filter and store at 4°C

Procedure

  • Inoculate 250 mL of SOB medium with 1 mL vial of seed stock and grow at 20°C to an OD600nm of 0.3.
    • This takes approximately 16 hours.
    • Room temperature will work. You can adjust this temperature somewhat to fit your schedule.
    • Aim for lower, not higher OD if you can't hit this mark.
  • Pre-chill in ice as many centrifuge tubes as needed.
  • Transfer the culture to the chilled centrifuge tubes.
  • Centrifuge at 3000g at 4°C for 10 minutes.
  • Discard supernatant.
  • Add 40mL of ice cold CCMB80 buffer first to resuspend the cells.
  • When cells are in suspension, add another 40mL of CCMB80 buffer for a total of 80mL.
  • Pipet buffer against the wall of the centrifuge bottle to resuspend cells.
    • Avoid pipetting directly into cell pellets.
  • Incubate on ice for 20 minutes.
  • Centrifuge at 3000G at 4°C for 10 minutes.
  • Discard supernatant.
  • Resuspend cell pellet in 10 mL of ice cold CCMB80 buffer.
    • If using multiple centrifuge tubes, combine the cells post-resuspension.
  • Incubate on ice for 20 minutes. Prepare for aliquoting
  • Aliquot 200 µL into chilled PCR tubes.
  • Store at -80°C indefinitely.
    • Note: Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency and thus leftover cells must be discarded.

Transformation

Procedure

  • Thaw a tube of competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µL of cells into a 1.5 mL microcentrifuge tube on ice.
  • 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.
  • Place the mixture on ice for 30 minutes. Do not mix.
  • Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  • Place on ice for 5 minutes. Do not mix.
  • Pipette 950 µL of room temperature SOC into the mixture.
  • Place at 37°C for 60 minutes shaking at 250 RPM.
  • Prepare LB Agar selection plates with the adequate antibiotic.
  • Spread 100 µL of the culture onto a selection plate and incubate overnight at 37°C.
    • Dilute prior to plating if necessary.

Miniprep

Isolation and propagation of DNA molecules are essential features of recombinant DNA technologies. To this purpose, DNA molecules are copied (propagated) using the replication machinery of its host. A plasmid or a vector is a circular DNA molecule that contains the information necessary for its replication inside a host. Plasmids and vectors contain antibiotic resistance genes allowing selection of bacteria hosting the plasmid of interest when a population of bacteria is cultured in the presence of the antibiotic. Plasmids and vectors also contain specific DNA sequences recognized by restriction enzymes allowing the insertion of DNA fragments through the combination of a host plasmid cut by a set of restriction enzymes and a DNA fragment with compatible ends in a reaction mediated by a ligase. The newly ligated plasmid containing the DNA insert will be transformed into a host to replicate and propagate itself. This host-assisted heterologous-DNA fragment replication process is known as cloning.

Materials

  • Solution I
    • Tris HCl 25 mM
    • EDTA 10 mM
  • Solution II
    • NaOH 0.2 N
    • SDS 41.9 mM
      • SDS must be dissolved carefully.
  • Solution III
    • Potassium acetate 3 M
      • Must be previously dissolved in water.
    • Acetic Acid 2 M
  • 1.5 mL microcentrifuge tubes
  • Isopropanol
  • Ethanol 70%
  • Nuclease-Free Water
  • RNAse

Before You Begin

Add 10 mL of LB media to a 50mL tube with the corresponding antibiotic, inoculate with a single colony from a plate. All cultures were incubated overnight at 37ºC and shaken at 250 rpm.

Procedure

  • Centrifuge for 5 min at 5000xg.
  • Discard supernatant.
  • Add 200 μL of Solution I and 1 μL of RNAse.
  • Resuspend until the pellet is completely solubilized.
  • Incubate 5 min at room temperature.
  • Add 200 μL of Solution II.
  • Mix by inversion.
  • Incubate 5 min at room temperature.
  • Add 200 μL of Solution III.
  • IMix by inversion.
  • Incubate 10 min at -20ºC.
  • Centrifuge for 10 min at 16000xg (14,000 RPM).
  • Transfer supernatant to a new 1.5 mL tube.
    • Avoid disturbing/touching white precipitate.
  • Add 500 μL of isopropanol.
  • Centrifuge for 10 min at 16000xg (14,000 RPM).
  • Discard supernatant.
  • Add 1 mL of ethanol 70%.
  • Mix by vortexing.
  • Centrifuge for 5 min at 16000xg (14,000 RPM).
  • Discard supernatant.
  • Dry tubes with open cap at 37ºC for 45 min.
  • Add 50 μL of Nuclease-Free water.
  • Mix by vortexing.
  • Storage at 4ºC for short periods of time (days) or -20ºC indefinitely.

Colony PCR

Materials

  • PCR tubes
  • Nuclease-Free water
  • Sterile loop

Procedure

  • Add 10-20 µL of Nuclease-Free water into a PCR tube.
  • Pick a single colony with a sterile loop.
  • Inoculate the water with the loop, making sure most bacteria ends in the water.
    • The water must become somewhat cloudy.
  • Incubate at 95°C for 10 minutes.
    • This could be easily done in a thermocycler.
  • Perform a usual PCR procedure using 1-2 µL of the lysed bacteria as template.

IPTG Induction

Procedure

  • Transform expression plasmid into E.coli BL21(DE3).
  • Plate on antibiotic selection plates and incubate overnight at 37°C.
  • Resuspend a single colony in 10 mL liquid culture with proper antibiotic.
  • Incubate at 37°C until OD600 reaches 0.4–0.8.
  • Induce with 10 µL of a 1 M stock of IPTG (final concentration of 1 mM) and induce for 2-4 hours at 37°C.
  • Analyze by method of choice.