• Add 20 µL sterile H2O to desired well in distribution plate.
• Incubate at room temperature for ~10 minutes.
• Transfer resuspension to microcentrifuge tube.

Materials

• Competent Cells
• DNA template
• 800 µL LB
• LB+Antibiotic Plates

Procedure

• Thaw competent cells on ice.
• Transfer 50 µL of competent cells to chilled falcon tubes.
• Add 1 µL of template to cells (2.5 µL if dilute).
• Incubate on ice for 30 minutes.
• Heat schock in 42 °C water bath for 90 seconds.
• Immediately place back onto ice for 2 minutes.
• Add 800 µL of LB to each tube.
• Incubate at 37 °C for 1 hour.
• Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.
• Incubate overnight at 37 °C.

Materials

• 22 µL dH20
• 1 µL BSA
• 5 µLBuffer
• 20 µL Template
• 1 µL Enzyme 1
• 1 µL Enzyme 2

Procedure

• Mix reactants, adding enzyme last.
• Place at 37 °C for 3-5 hours.
• If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
• Run on a gel and extract product.

Materials

• 10 uL Q Solution
• 5 µL 10x Buffer
• 1.25 µL 10mM dNTPs
• 1 µL Template
• 1 µL Forward Primer
• 1 µL Reverse Primer
• 0.3 µL Taq
• 0.15 µL PFU
• 30 µL dH20

Procedure

• Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
• Run in thermal cycler.

Materials

• 10 µL Q Solution
• 5 µL 10x Buffer
• 1.25 µL 10mM dNTPs
• 2.5 µL Forward Primer
• 2.5 µL Reverse Primer
• 0.3 µL Taq
• 0.15 µL PFU
• Template based on concentrations determined by SOEing calculator: “Link”
• ±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”

Procedure

• Mix reactants into PCR tubes.
• Run in thermal cycler.
• Continue PCR SOEing of parts until completed.

Materials

• Digested Vector
• Digested Insert
• Water
• T4 DNA Ligase
• T4 DNA Ligase Buffer

Procedure

• Mix these materials in the amounts determined by the reaction volume calculator here.

Materials

• 34 mM NaH₂PO₄
• 64 mM K₂HPO₄
• 20 mM (NH₄)₂SO₄
• 1 µM FeSO₄
• 0.1 mM MgSO₄
• 10 µM CaCl₂
• 30 mM Ethylene Glycol
• 30 mM Glycolate
• 0.5% wt/vol Casein Acid Hydrolysate
• 1.5% wt/vol Agar (if making solid media)

Procedure

• Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
• Mix together NaH₂PO₄, K₂HPO₄, (NH₄)₂SO₄, FeSO₄, MgSO₄, and CaCl₂.
• Titrate to pH 7 with HCl.
• Add the glycolate and casein acid hydrolysate.
• Mix in the ethylene glycol in a fume hood.
• If making solid media, also mix in agar.
• Autoclave on an appropriate cycle.

Materials

• Luria Broth Media
• Ethylene Glycol
• Tecan M200 Pro
• E. coli

Procedure

• Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
• Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
• Incubate at 37°C overnight on a shaker at 150 rpm.
• From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
• Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
• Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
• Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
• With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
• Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
• Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
• Aliquot the remaining dilutions as the diagram below depicts.
• Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
• Follow the steps of your Tecan machine appropriate to the specific bacterial strain.

Materials

• Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
  • K₂HPO₄: 10.5g
  • KH₂PO₄: 4.5g
  • (NH₄)₂SO₄: 1g
  • Sodium Citrate * 2H₂O: 0.5g
  • 1M Tris (pH 7.5): 200mL Tris

• EMS (Sigma)
• 50mL Conical Corning tubes
• 15mL Falcon tubes
• LB Broth
• Ethylene Glycol Agar Plates
• Serological pipets (10mL) for cell re-suspension
• PipetAide

Procedure

• Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD~0.2.
• Chill the cells on ice and spin down the 10mL aliquots at max speed in Eppendorf Centrifuge 5810 R for 10 minutes.
• Wash twice with 10mL Buffer A.
• Pipet up and down to re-suspend pellet
• Pellet cells as in step 2
• Decant supernatant in waste
• Bleach waste