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Team:NTNU Trondheim/Protocols
From 2014.igem.org
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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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Revision as of 21:19, 17 October 2014
Team:NTNU Trondheim/Protocols
From 2014.igem.org
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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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Media
Techniques
Plasmids
Calculations
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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
{{{tech}}}
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
{{{tech}}}
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
{{{tech}}}
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
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.
DNA isolation and cleaning
{{{tech}}}
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
{{{tech}}}
DNA digests for both ligation and verification used the protocol in the PhotoSynLab wiki.
PCR
{{{tech}}}
The touchdown PCR procedure detailed on the PhotoSynLab wiki was used to amplify DNA.
Nanodrop
{{{tech}}}
A NanoDrop ND-1000 Spectrophotometer was used to determine DNA concentrations.
3A assembly
{{{tech}}}
3A assembly was conducted according to the iGEM 3A assembly protocol.
Ligation
{{{tech}}}
Ligation was performed according to the protocol outlined on the PhotoSynLab wiki.
Transformation (Escherichia coli)
{{{tech}}}
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)
{{{tech}}}
The transformation procedure for transforming Synechocystis can be found at the PhotoSynLab wiki.
Right flank
{{{tech}}}
Left flank
{{{tech}}}
Kanamycin resistance
{{{tech}}}
Lac promotor
{{{tech}}}
Glucose oxidase
{{{tech}}}
Left flank + Kanamycin resistance + Right flank
{{{tech}}}
DNA concentration
{{{tech}}}
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
