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Team:UC Davis/Notebook/Protocols
From 2012.igem.org
Protocols
•Add 20ul 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 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.
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.
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.
Coming soon.
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.
