Team:Pittsburgh/Protocols

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Office of the Provost Department of Bioengineering Department of Biological Sciences Department of Chemistry Swanson School of Engineering
IDT NEB Experiment

The protocols we reference in our Notebook

Contents

Reporter

Pouring Plates

  1. In 1L of DI water, add 25 grams of LB broth and 15 grams of agar. Adjust accordingly if making a different volume
  2. Swirl gently to dissolve some of the LB broth
  3. Autoclave for 20 minutes
  4. Remove from the autoclave and let cool
  5. When cool, add antibiotic to working concentration
    • Ampicillin - 100 µg/mL
    • Chloramphenicol - 25 µg/mL
    • Kanamycin - 50 µg/mL
  6. Pour plates
  7. Let them sit for ~30 min to solidify
  8. Store in cold room in original plate sleeve. Make sure agar side is up
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Competent Cells

We follow the protocol for the preparation of competent E. coli cells using calcium chloride from Ivaan.com. We use the Competent Cell Test Kit from iGEM to determine our cells' competency.

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Transformations

We follow the transformation protocol provided by iGEM. Any deviations from this protocol are noted in the Lab Notebook.

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Liquid Cultures

  1. In a 15 mL centrifuge tube, add 5 mL of LB Broth
  2. Add necessary antibiotic to working concentration
  3. Using a pipette tip, lift colony of interest
  4. Place tip in centrifuge tube
  5. Tape on the lid. Be sure not to twist tightly
  6. Incubate at 37°C in the shaker overnight
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Glycerol Stocks

  1. Combine 1 mL of 40% glycerol with 1 mL fresh bacterial culture in a cryogenic vial
  2. Gently vortex or pipette to mix
  3. Store culture in -80°C
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Minipreps

We follow the miniprep protocol provided by Thermo Scientific for their GeneJET Plasmid Miniprep Kit. We elute the DNA in 25 µL of Elution Buffer, not 50 µL as stated in the protocol, to achieve a higher concentration.

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Sequencing

We use Genewiz for sequencing and follow their sample submission guidelines.

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Restriction Digests

We use the protocol provided by Thermo Scientific for the fast digestion of DNA as a guide.

  1. For a 20 µL reaction, add:
    • 2 µL enzyme (1 µL each if doing a double digest)
    • 2 µL 10x buffer
    • 1 µg plasmid
    • Nuclease-free water to volume
  2. Incubate at 37°C for 30 min
  3. Incubate at 65°C for 20 min to deactivate enzymes
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Agarose Gel Electrophoresis

All gels are 1% agarose unless otherwise stated. We use the 1 kb DNA ladder from NEB.

  1. Dissolve 0.5 g agarose in 50 mL 1X TAE buffer, using the microwave to heat
  2. When cool to the touch, add 5 µL ethidium bromide and pour gel
  3. Run in 1X TAE buffer for 45 min
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Gel Extraction and DNA Cleanup

We follow the protocols provided by Thermo Scientific for their GeneJET Gel Extraction and DNA Cleanup Micro Kit.

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Dephosphorylation

  1. Combine
    • 10 µL DNA
    • 1 µL buffer
    • 0.5 µL rSAP (recombinant shrimp alkaline phosphatase)
  2. Incubate at 37°C for 30 min
  3. Incubate at 65°C for 5 min to deactivate enzymes
  4. Transform 5 uL of each reaction
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Ligation

  1. Combine
    • 1 µL plasmid
    • amount of insert for desired ratio
    • 2 µL buffer
    • Nuclease-free water to 20 µL
    • 1 µL T4 DNA ligase
  2. Incubate at room temperature for 10 min
  3. Incubate at 65°C for 10 min to deactivate enzymes
  4. Transform 5 µL of each reaction
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Polymerase Chain Reaction

We follow the PCR protocol provided by NEB for Phusion High-Fidelity DNA Polymerase.

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Colony PCR

We use the Colony PCR Protocol for plated colonies from the 2013 iGEM page.

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Site-Directed Mutagenesis

  1. Phosphorylate Oligos
    • T4 PNK 1 µl (10 units)
    • 10X T4 Ligase buffer 5 µl
    • DNA (20 mer) 1-2 µg
    • Nuclease-free water to 50 µl
    • Incubation 37°C for 30 minutes
  2. Mix in a PCR tube
    • 0.5 µl forward primer, 2.5 pmoles/µl
    • 0.5 µl reverse primer, 2.5 pmoles/µl
    • 0.25 µl 40 mM dNTP mix, (10 mM each)
    • 1.25 µl Phusion Buffer
    • 1 µl Template DNA, 2 ng/µl
    • 0.25 ul Phusion
    • 8.75 µl sterile H2O
    • 12.5 µl total
  3. Incubate according to the following PCR Program:
    1. 5 min at 95°C
    2. Repeat 18 times:
      1. 50 s at 95°C
      2. 50 s at 60°C
      3. 1 min + 1 min/1 kb template at 68 °C
    3. 7 min at 68 °C
  4. Gel Check: Run 2.5 µl of the reaction on a gel. There should be a band corresponding to your product. Even if you don’t see a reaction product, you can still try the rest of the protocol, but you may not get any colonies.
  5. DpnI Digest: Add 0.25 µl of DpnI (20 U/µl, New England Biolabs) to the reaction. Incubate at 37◦C for 1 hr.
  6. Transform
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Annealing

Phosphorylation

  1. Resuspend oligos to a stock concentration of 100µM with nuclease-free water.
  2. To a PCR tube, add
    • 2 µl of the proper Top or Bottom strand oligo
    • 2 µl of 10X T4 DNA Ligase Buffer
    • 1 µl of T4 Polynucleotide Kinase
    • 15 µl of water
  3. Mix well and spin down. Oligo final concentration is 10 uM.
  4. Incubate the PCR tube in the thermocycler with the program at 37°C for 60 minutes, then at 65°C for 20 minutes then end.
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10X Annealing Buffer

For 4 mL, add

  • 400 µL 1M Tris pH 8
  • 80 µL 0.5M EDTA pH 8
  • 800 µL 2.5M NaCl
  • 2720 µL water
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Annealing in Buffer

  1. Combine
    • 5 µL forward oligo
    • 5 µL reverse oligo
    • 5 µL 10X annealing buffer
    • 35 µL water
  2. Mix well and spin down. The final oligo concentration is 1µM.
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Annealing in Water

  1. Combine
    • 1 µg oligos
    • 5 µL 10X T4 DNA Ligase Buffer
    • Nuclease-free water to 50 µL
  2. Incubate at 85°C for 10 min
  3. Cool to 20°C over 30 min
  4. Store annealed oligos at -20°C
Any deviations from this protocol are noted in the Lab Notebook.
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Cell-Free Expression

We follow the protocol provided by NEB for the protein synthesis reaction using PURExpress (E6800).

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DNAzyme

10X TBE Buffer

For 1 liter, combine

  • 108 g Tris base
  • 55 g boric acid
  • 40 mL 0.5 M EDTA (pH = 8.0)
  • water to 1 liter
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Denaturing Polyacrylamide Gel Electrophoresis

We use Novex precast gels in TBE buffer for our dPAGE assays. We follow the guidelines provided with the gel loading buffer we use from ThermoFisher. We use SYBR Gold from ThermoFisher to stain.

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Native Polyacrylamide Gel Electrophoresis

  1. For a 15% gel, combine
    • 4 mL 40% acrylamide
    • 6 mL 1X TBE buffer
    • 100 µL 10% APS
    • 15 uL TEMED
    in a 15-mL Falcon tube
  2. Invert tube to mix
  3. Pour between plates and insert comb
  4. Gel solidifies in about 20 minutes
  5. Load gel and run at 100V for one hour
  6. Stain for 20 minutes with SYBR Gold from ThermoFisher

Thank you to Nick from the Deiters lab for his help!

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Buffer B

Combine

  • 25 mM NaCl
  • 50 mM MOPS
  • NaOH to adjust pH to 7.5
  • water to volume
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Cleavage Reaction

  1. Combine
    • 0.7 μM annealed duplex
    • 10 μM ErCl3 dissolved in water
    • Buffer B to volume
  2. Incubate at room temperature for 20 minutes
  3. Quench with equal volume of gel loading buffer II from ThermoFisher
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    Collaborations

    InterLab Study

    We followed the updated Plate Reader Protocol from iGEM.

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    1X PBS

    • For 1 liter, dissolve the following in 800 mL of water
      • 8 g NaCl
      • 0.2 g KCl
      • 1.44 g Na2HPO4
      • 0.24 g KH2PO4
    • Adjust pH to 7.4 using NaOH
    • Add water to volume
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    University of Georgia: Archaeal InterLab

    We were given five samples from UGA. 50 μL of sample was added to a 96-well plate for each triplicate of each sample. Fluorescence was read at 590 nm excitation, 645 nm emission, and 630 nm cutoff.

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    William & Mary: Genetic Circuits Toolbox

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    50X TAE Buffer

    Materials

    • 242 grams Tris free base
    • 18.61 grams Disodium EDTA
    • 57.1 mL glacial acetic acid
    • DI water

    Procedure

    1. Add Tris free base and EDTA to ~700 mL of DI water
    2. Stir until dissolved
    3. Autoclave for 20 minutes
    4. Add acetic acid
    5. Adjust the volume with DI water until the volume is 1 L
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