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Revision as of 17:37, 23 August 2012

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Protocols

These are our protocols.

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

Transformations

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.

Restriction Enzyme Double Digest

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.

PCR (sequence dependent)

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.

PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)

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.

Ligation

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.

Ethylene Glycol Media

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.

Ethylene Glycol Toxicity Assay

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