Team:NTNU Trondheim/Protocols
From 2014.igem.org
Team:NTNU Trondheim/Protocols
From 2014.igem.org
Team:NTNU_Trondheim/Protocols
From 2014.igem.org
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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)
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Plasmids
- Right flank
- Left flank
- Kanamycin
- Lac inducible promoter
- Glucose oxidase
- Left flank + kan + right flank
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Organisms
- Escherichia coli DH5α
- Synechocystis sp. PCC 6803
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Calculations
- 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
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Made LB plates with ampicillin and ampicillin + kanamycin.
yB medium
Recipe
Ingredients:
- Yeast extract (2.5g)
- Bactotryptone (10g)
- KCL (0.38g)
- Fill with 0.5 L of distilled / filtered H2O.
- Add KOH until the pH is 7.4, then autoclave at 121 °C for 20 minutes.
- Add 17 mL sterile 1M MgSO4
Synechocystis medium
Recipe
To grow Synechocystis cultures, BG-11 medium was made according to the recipe found on the PhotoSynLab wiki.
Gibson Assembly
Protocol from New England Biolabs
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- 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.
DNA isolation and cleaning
For plasmid isolation, the Promega Wizard Plus SV Minipreps DNA Purification System A1460 Miniprep protocol was used. For PCR product isolation, the QIAquick PCR Purification kit was used.
DNA digestion
DNA digests for both ligation and verification used the protocol in the PhotoSynLab wiki.
PCR
The touchdown PCR procedure detailed on the PhotoSynLab wiki was used to amplify DNA.
Nanodrop
A NanoDrop ND-1000 Spectrophotometer was used to determine DNA concentrations.
3A assembly
3A assembly was conducted according to the iGEM 3A assembly protocol.
Ligation
Ligation was performed according to the protocol outlined on the PhotoSynLab wiki.
Transformation (Escherichia coli)
The protocols used for preparing competent DH5a cells, as well as the heat-shock transformation procedure employed can be found at the PhotoSynLab wiki.
Transformation (Synechocystis sp. PCC 6803)
The transformation procedure for transforming Synechocystis can be found at the PhotoSynLab wiki.