Team:NTNU Trondheim/Protocols

From 2014.igem.org

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<h3 class="centered">Gibson Assembly</h3>
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<div class="entry pc-techniques">
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<h6>Protocol from New England Biolabs
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<div class="nb-onetech-i nohilite">show technical details</div>
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<div class="nb-tech">{{{tech}}}</div>
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<br><li>Set up the following reaction on ice:</li> <br>
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                <td rowspan="2">&nbsp;</td>
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                <td colspan="3" align="center">Recommended Amount of Fragments Used for Assembly</td>
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            <tr>
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                <td align="center">2-3 Fragment Assembly</td>
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                <td align="center">4-6 Fragment Assembly</td>
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                <td align="center">Positive Control**</td>
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                <td>Total Amount of Fragments</td>
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                <td align="right">0.02–0.5 pmols*<br>
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                X μl</td>
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                <td align="right">0.2–1 pmols*<br>
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                X μl</td>
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                <td align="center">10 μl</td>
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                <td>Gibson Assembly Master Mix (2X)</td>
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                <td align="right">10 μl</td>
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                <td align="right">10 μl</td>
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                <td align="center">10 μl</td>
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                <td>Deionized H<sub>2</sub>O</td>
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                <td align="right">10-X μl</td>
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                <td align="right">10-X μl</td>
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                <td align="center">0</td>
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                <td>Total Volume</td>
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                <td align="right">20 μL***</td>
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                <td align="right">20 μL***</td>
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                <td align="center">20 μL</td>
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    * 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>
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** Control reagents are provided for 5 experiments.<br>
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*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
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<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>
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<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>
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</li><br>
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<li>
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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.
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Revision as of 15:15, 14 October 2014

Team:NTNU Trondheim/Protocols - 2014.igem.org

 

Team:NTNU Trondheim/Protocols

From 2014.igem.org

Team:NTNU_Trondheim/Protocols - 2014.igem.org

 

Team:NTNU_Trondheim/Protocols

From 2014.igem.org

NTNU Genetically Engineered Machines

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
Chloramphenicol30 mg / mL30 μg / mL 1000Ethanol-20 °C
Kanamycin50 mg / mL30 μg / mL1000Filter sterilized H2O4 °C
Spectinomycin50 mg / mL50 μg / mL1000Filter sterilized H2O4 °C

Ingredients:

  • Tryptone (10g)
  • NaCl (10g)
  • Yeast Extract (5g)

  1. Fill with 1 L of distilled / filtered H2O.
  2. Autoclave at 121 °C for 20 minutes.
  3. 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}}}


  1. Set up the following reaction on ice:

    2.   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
    * 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%.
    ** Control reagents are provided for 5 experiments.
    *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.


  2. 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).

  3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.

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