Team:Exeter/Protocols

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Exeter | ERASE

Contents

Protocols

Restriction Digestion

Materials

  • Part A (Purified DNA to be digested, > 16ng/ul)
  • Part B (Purified DNA to be digested, > 16ng/ul)
  • dH2O (to fill up to 16ul total volume)
  • NEB Buffer 2 (5ul)
  • BSA (1ul) Restriction
  • Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
  • Linearized plasmid backbone (pSB1C3, 25ng/ul)

Method

  1. Label two 0.6ml thin-walled tubes A and B.
  2. Add 250ng of DNA samples A and B to be digested into their corresponding tube and adjust with dH2O for a total volume of 16ul.
  3. Add 2.5ul of NEBuffer 2 0.and 5ul of BSA to each tube,
  4. In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
  5. In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
  6. In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
  7. Incubate the restriction digest at 37oC for 30min, and then 80oC for 20min to heat kill the enzymes.

Ligation

Materials:

  • Part A (EcoRI-HF SpeI) digested DNA fragment, ( < 3ul)
  • Part B (XbaI PstI )digested DNA fragment ( < 3ul)
  • Digested pSB1C3 plasmid backbone (25ng/ul)
  • dH2O (to fill up to 10ul total volume)
  • T4 DNA ligase buffer
  • T4 DNA ligase

Method:

  1. Add 2ul of digested pSB1C3 plasmid backbone from the digestion procedure (25 ng)
  2. Add equimolar amount of digested Part A (< 3 ul)
  3. Add equimolar amount of digested Part B (< 3 ul)
  4. Add 1 ul T4 DNA ligase buffer.
  5. Add 0.5 ul T4 DNA ligase
  6. Add water to fill up to a volume of 10 ul
  7. Ligate at 16oC/30oC for 30min, and then 80oC for 20min to heat kill the enzymes.

Transformation

Materials:

  • Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)
  • Competent cells (DH5α and TOP10)
  • Ice
  • 2ml tubes
  • SOC media
  • 42ºC water bath
  • Petri dishes with LB agar and appropriate antibiotic (Either ampicillin or Chloramphenicol)
  • Plastic spreader
  • 37ºC shaking and standard incubators
  • 10pg/ul RFP Control (pSB1A3 w/ BBa_J04450)

Method:

  1. Add 50 µL of thawed competent cells pre-chilled 2ml tubes for each part and another 50µL into a 2ml tube labelled as a control.
  2. Add 1 - 2 µL of the resuspended DNA to each 2ml tube and pipet it up and down gently.
  3. Add 1 µL of the RFP Control (pSB1A3 w/ BBa_J04450) to the control transformation.
  4. Incubate cells on ice for 25 minutes and then perform heat shock on the cells by immersion in a 42ºC pre-heated water bath for 45 seconds.
  5. Once removed, incubate the cells on ice for 5 minutes.
  6. Add 200 μl of SOC media to each transformation and incubate the cells at 37ºC for 2 hours in a shaking incubator.
  7. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
  8. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
  9. Incubate the plates at 37ºC for 12-14 hours overnightin a standard incubator.

Colony Picking

Materials:

  • Liquid lysogeny broth Agar
  • Transformed plated cells, incubated at 37°C overnight
  • Relevant Antibiotic (AMP or CAM)
  • Shaking incubator at 37°C

Method:

  1. To a 5ml Liquid lysogeny broth Agar culture add 5 ul of the relevant antibiotic to form the inoculum solution.
  2. Pick a single, freshly grown colony with a pipette tip and inoculate the solution.
  3. Cultivate the inoculum for 16 hours in the shaking incubator at 37°C.

Glycerol Stock Preparation

Materials:

  • Sterile 80% glycerol solution
  • Inoculum containing colonies incubated for 16 hours in in the shaking incubator at 37°C
  • Vortexer
  • Centrifuge
  • -80°C Freezer

Method:

  1. 0.5ml of this culture inoculated into sterile vial.
  2. Add 0.5ml of 80% glycerol to the tube and vortex to mix.
  3. Centrifuge at 8000 rpm (6800 x g) for 20 seconds.
  4. Store glycerol stock in freezer at -80°C for future use.

Miniprep

Materials:

  • Eppendorf tubes
  • Thermo Scientific GeneJET Plasmid Miniprep Kit:
    • Resuspension Solution
    • Lysis Solution
    • Neutralization Solution
    • Wash Solution
    • Elution Buffer (Not used)
    • Thermo Scientific GeneJET Spin Column
  • dH2O (In place of elution buffer)
  • Microcentrifuge
  • Vortexer

Method:

  1. Place the transformed bacterial culture into an Eppendorf tube and harvest the transformed bacterial culture by centrifugation at 8000 rpm (6800 x g) in a Microcentrifuge for 2 minutes at room temperature.
  2. Decant the supernatant and remove all remaining medium.
  3. Resuspend the pelleted cells with 250 ul Resuspension solution then vortex the solution.
  4. Add 250 ul of Lysis solution and invert the tube 4-6 times.
  5. Add 350 ul of Neutralization solution to inhibit the Lysis solution and invert the tube 4-6 times. Centrifuge for 5 minutes at 10,000-14,000g (≥ 12,000 x g).
  6. Transfer the supernatant to the Thermo Scientific GeneJET Spin Column and centrifuge for 1 minute at 10,000-14,000g (≥ 12,000 x g).
  7. Wash the column by adding 500ul of Wash Solution and centrifuge for 30-60 seconds. This was repeated twice.
  8. Transfer the column to a new Eppendorf tube and add 50ul of dH2O to elute the DNA. Incubate this for 2 minutes before centrifuging for 2 minutes at 10,000-14,000g (≥ 12,000 x g).
  9. Collect the purified DNA in the flow-through.

LB Media

Used to grow overnight cultures of E. coli.
The recipe for 1l LB media is as follows:
Tryptone15g
Yeast Extract10g
NaCl                       5g
Once these components have been added ddH2O should be used to bring the solution to 1l, followed by autoclaving.

MYE Media

Exeter | ERASE

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