3.1.11: Linearizing Backbone Supplies

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Page ID: 67618

Nathan Reyna, Ruth Plymale, & Kristen Johnson
Ouachita Babtist University & University of New Hampshire

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Linearizing Backbones:

Materials	Amount Needed: (Per Backbone Reaction)
Q5 High-Fidelity 2X Master Mix (Cat #: M0492S)	Up to 25 uL
10 uM Forward Primer (SB-prep-3P-1)	Up to 2.5 uL
10 uM Reverse Primer (SB-prep-2Ea)	Up to 2.5 uL
Template DNA (psB1C3, A3, K3, T3)	10 ng
Distilled Water	Up to 60 uL
Thermocycler	1
UV Spectrophotometer	1
Micropipette and Tips	1-10 uL, 0.2-1 uL, 20-200 uL
Heat Block (80C)	1
Gel Rig	1
PCR Clean-up Kit (Cat #: D4033)	1
10X NEB Buffer 2.1	4 uL
Digestion Enzymes (EcoRI, PstI, DpnI)	1 uL of each
Competent Bacteria Cells (JM109= Cat#: T3003)	25 uL
LB Agar Plate with Antibiotic (Chlor, Amp, Tet, Kan)	1 plate (depending on backbone)
Sterile Glass Beads	Approximately 4-8 beads
SOC Media (Cat#: 15544034)	200 uL
Shaking Incubator (37C)	1
T4 DNA ligase (Cat#: M0202S)	0.5 uL
T4 DNA Ligase Buffer(Cat#: B0202S)	1 uL