Team:NYU Shanghai/Protocols
We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly.
50mL LB + 500µL Arabinose* + 50µL Ampicillin**
*2mg/mL of Arabinose Stock Solution
**100mg/mL of Ampicillin Stock Solution
Total Amount of Reagent | 100mL | 250mL |
Deionized Water | 100mL | 250mL |
Yeast | 1g | 2.5g |
Tryptone | 0.5g | 1.25g |
NaCl | 1g | 2.5g |
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 |
General Overview:
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 |
Estimated time: 3 hours (plus 14-18 hour incubation)
Materials
Estimated time
Materials
Link to NEB Protocol
To determine buffer for double digests
Guide to heat inactivation
General reaction set-up:
Restriction enzyme | 10units, generally 1uL |
DNA | 1ug |
10X NEB Buffer | 5uL (1X) |
Total Reaction Volume | 50uL |
Incubation Time | 1hr |
Incubation Temperature | Enzyme Dependent |
Estimated time:
Materials
MinElute Midi Gel Extraction Kit
Procedure