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Revision as of 18:52, 3 August 2012

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Protocols

These are our protocols.

Registry Part Distribution Rehydration
•Add 20ul 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 ul LB
•LB+Antibiotic Plates

Procedure

•Thaw competent cells on ice.
•Transfer 50 uL of competent cells to chilled falcon tubes.
•Add 1 uL of template to cells (2.5 uL 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 uL of LB to each tube.
•Incubate at 37 °C for 1 hour.
•Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
•Incubate overnight at 37 °C.

Restriction Enzyme Double Digest

Materials

•22 uL dH20
•1 uL BSA
•5 ul Buffer
•20 uL Template
•1 uL Enzyme 1
•1 uL 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 uL 10x Buffer
•1.25 uL 10mM dNTPs
•1 uL Template
•1 uL Forward Primer
•1 uL Reverse Primer
•0.3 uL Taq
•0.15 uL PFU
•30 uL 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.

SOEing
Coming soon.

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.