Team:NTNU Trondheim/Protocols
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- | <p> | + | <p> <p> Ingredients (1L): <br> <ul> <li>Bactotryptone (20 g)</li> <li>Yeast extract (5g)</li> <li>NaCl (0.584g)</li> <li>KCl (0.186g)</li> <li>Agar (20g ONLY IF MAKING PLATES)</li> </ul> <br> <p> <ol> <li>Fill with 0.5 L of distilled / filtered H<sub>2</sub>O.</li> <li>Autoclave at 121 °C for 20 minutes. </li> <li>Add 10 mL 1M MgCl<sub>2</sub>, 10 mL MgSO<sub>4</sub> and 20 mL 1M glucose (all should be sterile) prior to use </li> |
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- | + | The Gibson assembly protocol can be found at <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">New England Biolabs</a>. | |
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Latest revision as of 21:39, 17 October 2014
Team:NTNU Trondheim/Protocols
From 2014.igem.org
Team:NTNU_Trondheim/Protocols
From 2014.igem.org
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Media
- Lysogeny Broth (LB)
- SOC medium
- 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)
-
Plasmids
- Right flank
- Left flank
- Kanamycin resistance
- Lac promoter
- Glucose oxidase
- Left flank + Kan + Right flank
-
Calculations
- DNA concentration
Protocols
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![](https://static.igem.org/mediawiki/2014/4/4d/NTNU_media_on.png)
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Lysogeny 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.
SOC medium
Recipe
show technical details
Ingredients (1L):
- Bactotryptone (20 g)
- Yeast extract (5g)
- NaCl (0.584g)
- KCl (0.186g)
- Agar (20g ONLY IF MAKING PLATES)
- Fill with 0.5 L of distilled / filtered H2O.
- Autoclave at 121 °C for 20 minutes.
- Add 10 mL 1M MgCl2, 10 mL MgSO4 and 20 mL 1M glucose (all should be sterile) prior to use
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
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.
Right flank
Left flank
Kanamycin resistance
Lac promotor
Glucose oxidase
Left flank + Kanamycin resistance + Right flank
DNA concentration
After measuring the concentration of a sample of DNA in ng/ul with the NanoDrop method, the concentration in terms of pmolar could be determined with the equation