Team:NYU Shanghai/Protocols
50mL LB + 500µL Arabinose* + 50µL Ampicillin**
*2mg/mL of Arabinose Stock Solution
**100mg/mL of Ampicillin Stock Solution
Total Amount of Reagent: | 100 mL | 250 mL |
Deionized Water | 100 mL | 250 mL |
Yeast | 1 g | 2.5 g |
Tryptone | 0.5 g | 1.25 g |
NaCl | 1 g | 2.5 g |
Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.
SOB medium is also available dry premixed from Difco, 0443-17.
Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.
15/10 medium
Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:
Reagent | 50 mL (in Mols) | 100 mL (in M) | 100 mL (in Grams) |
Tryptone | (2% w/v) 1 g | 2 g | 2 g |
Yeast Extract | (0.5% w/v) 0.25 g | 0.5 g | 0.5 g |
NaCl | 0.0005 M | 0.01 mol | 0.0584g |
KCl | 0.000125 M | 0.0025 M | 0.0186 g |
MgCl2 | 0.0005 M | 0.01 M | 0.09521 g |
Glucose | 0.001 M | 0.02 M | 0.36032 g |
For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.
Reagent Solution:Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in polypropylene or glass.
Preparing 1mM D-Luciferin:Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4 uL of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.
Procedure:Chemical Name: | Tris-HCl | EDTA | NaCl |
Molecular Weight: | N/A | 292.23 g/mol | 58.44 g/mol |
Molarity Desired: | 10 mM | 1mM | 0.1M |
Calculation: | Dilute 1M Tris-HCl: | ||
Final Amount: | 150 uL (+14.85 mL ddH2O) | 0.00438 g | 0.08766 g |
Lysozyme Solubility: | 10 mg lysozyme in 1 mL of 10 mM Tris-HCl |
Desired Amount of Lysozyme Solution to Make: | 15 mL |
Amount of Lysozyme Needed: | 10 mg x 15 = 150 mg |
Amount of 10 mM Tris-HCl Needed: | 15 mL |
Chemical Name: | ATP disodium salt trihydrate | MgSO4•7H2O | HEPES Buffer | D-Luciferin free acid |
Molecular Weight: | 605.24 g/mol | 246.5 g/mol | 238.3 g/mol | 280.33 g/mol |
Molarity Desired: | 3 mM | 15 mM | 30 mM | 1 mM |
Calculation: | Use the 1 mM stock solution created earlier | |||
Final Amount: | 0.039945 g | 0.081345 g | 0.1573 g | 22 mL |
Link to NEB Protocol
To determine buffer for double digests, we used: NEB Protocol
Guide to heat inactivation: NEB Protocol
General reaction set-up:
Restriction enzyme | 10units, generally 1uL |
DNA | 1 ug |
10X NEB Buffer | 5uL (1X) |
Total Reaction Volume | 50uL |
Incubation Time | 1hr |
Incubation Temperature | Enzyme Dependent |