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Team:NTNU Trondheim/Protocols
From 2014.igem.org
Team:NTNU Trondheim/Protocols
From 2014.igem.org
"
Team:NTNU_Trondheim/Protocols
From 2014.igem.org
Jump to:
-
Media
- Liquid Broth (LB)
- Super Optimal Broth (S.O.B.)
- yB medium
- Synechocystis medium
-
Techniques
- Gibson assembly
- DNA isolation and cleaning
- DNA digestion
- PCR
- NanoDrop
- 3A assembly
- Ligation
- Transformation (Escherichia coli)
- Transformation (Synechocystis sp. PCC 6803)
- Gel electrophoresis
-
Plasmids
- Right flank
- Left flank
- Kanamycin
- Lac inducible promoter
- Glucose oxidase
-
Organisms
- Escherichia coli DH5α
- Synechocystis sp. PCC 6803
-
Calculations
- Transformation efficiency
- Enzyme amount
- DNA concentration
Protocols
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Liquid Broth (LB)
Recipe
Antibiotic additions
| Antibiotic | Stock concentration | Final concentration | Dillution factor | Solvent | Storage temperature |
|---|---|---|---|---|---|
| Ampicillin | 50 mg / mL | 50 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
| Chloramphenicol | 30 mg / mL | 30 μg / mL | 1000 | Ethanol | -20 °C |
| Kanamycin | 50 mg / mL | 30 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
| Spectinomycin | 50 mg / mL | 50 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
Ingredients:
- Tryptone (10g)
- NaCl (10g)
- Yeast Extract (5g)
- Fill with 1 L of distilled / filtered H2O.
- Autoclave at 121 °C for 20 minutes.
- Add antibiotics if needed, after the medium has cooled down.
Super Optimal Broth (S.O.B.)
Recipe
show technical details
{{{tech}}}
Made LB plates with ampicillin and ampicillin + kanamycin.
Gibson Assembly
Protocol from New England Biolabs
show technical details
{{{tech}}}
- Set up the following reaction on ice:
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section). - Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.
| Recommended Amount of Fragments Used for Assembly | |||
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
| Total Amount of Fragments | 0.02–0.5 pmols* X μl |
0.2–1 pmols* X μl |
10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μL*** | 20 μL*** | 20 μL |
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Gibson Assembly
Protocol from New England Biolabs
show technical details
{{{tech}}}
- Set up the following reaction on ice:
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section). - Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.
| Recommended Amount of Fragments Used for Assembly | |||
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
| Total Amount of Fragments | 0.02–0.5 pmols* X μl |
0.2–1 pmols* X μl |
10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μL*** | 20 μL*** | 20 μL |
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
