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Team:NTNU Trondheim/Protocols
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| + | <div class="twelve columns"> | ||
| + | <h3 class="centered">Gibson Assembly</h3> | ||
| + | </p> | ||
| + | <div class="entry pc-tech"> | ||
| + | <div> | ||
| + | <h6>Protocol from New England Biolabs | ||
| + | <div class="nb-onetech-i nohilite">show technical details</div> | ||
| + | </h6> | ||
| + | <div class="nb-tech">{{{tech}}}</div> | ||
| + | <p> | ||
| + | <ol> | ||
| + | <br><li>Set up the following reaction on ice:</li> <br> | ||
| + | </p> | ||
| + | <p> | ||
| + | <table width="100%" border="0" cellspacing="0" cellpadding="0" align="center"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td rowspan="2"> </td> | ||
| + | <td colspan="3" align="center">Recommended Amount of Fragments Used for Assembly</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td align="center">2-3 Fragment Assembly</td> | ||
| + | <td align="center">4-6 Fragment Assembly</td> | ||
| + | <td align="center">Positive Control**</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total Amount of Fragments</td> | ||
| + | <td align="right">0.02–0.5 pmols*<br> | ||
| + | X μl</td> | ||
| + | <td align="right">0.2–1 pmols*<br> | ||
| + | X μl</td> | ||
| + | <td align="center">10 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Gibson Assembly Master Mix (2X)</td> | ||
| + | <td align="right">10 μl</td> | ||
| + | <td align="right">10 μl</td> | ||
| + | <td align="center">10 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Deionized H<sub>2</sub>O</td> | ||
| + | <td align="right">10-X μl</td> | ||
| + | <td align="right">10-X μl</td> | ||
| + | <td align="center">0</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total Volume</td> | ||
| + | <td align="right">20 μL***</td> | ||
| + | <td align="right">20 μL***</td> | ||
| + | <td align="center">20 μL</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | * Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.<br> | ||
| + | ** Control reagents are provided for 5 experiments.<br> | ||
| + | *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required. | ||
| + | </p> | ||
| + | <br> | ||
| + | <li>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. <br><br> | ||
| + | <i>Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see <a href="https://www.neb.com/tools-and-resources/search?type=FAQ">FAQ</a> section).</i> | ||
| + | </li><br> | ||
| + | <li> | ||
| + | Transform NEB 5-alpha Competent <i>E. coli</i> cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol. | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <div id="week36entry" class="nb-week" style="display: block"> | ||
<div class="twelve columns"> | <div class="twelve columns"> | ||
<h3 class="centered">Gibson Assembly</h3> | <h3 class="centered">Gibson Assembly</h3> | ||
Revision as of 12:32, 8 August 2014
Team:NTNU Trondheim/Protocols
From 2014.igem.org
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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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Media
Techniques
Plasmids
Organisms
Calculations
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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.
