3.1.11: Linearizing Backbone Supplies
- Page ID
- 67618
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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 |