Team:NTNU Trondheim/Protocols

From 2014.igem.org

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<h6>Protocol from New England Biolabs
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<div class="nb-onetech-i nohilite">show technical details</div>
<div class="nb-onetech-i nohilite">show technical details</div>
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<div class="nb-tech">{{{tech}}}</div>
<div class="nb-tech">{{{tech}}}</div>
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<p>
 
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<br><li>Set up the following reaction on ice:</li> <br>
 
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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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                <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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                <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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<br>
 
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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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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 21:38, 17 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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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
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.

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)

  1. Fill with 0.5 L of distilled / filtered H2O.
  2. Autoclave at 121 °C for 20 minutes.
  3. 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)

  1. Fill with 0.5 L of distilled / filtered H2O.
  2. Add KOH until the pH is 7.4, then autoclave at 121 °C for 20 minutes.
  3. 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

show technical details
{{{tech}}}

The Gibson assembly protocol can be found at New England Biolabs.

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}}}

BBa_K1424001.

Left flank

{{{tech}}}

BBa_K1424000.

Kanamycin resistance

{{{tech}}}

BBa_K1424003.

Lac promotor

{{{tech}}}

BBa_K1424002.

Glucose oxidase

{{{tech}}}

BBa_K1424004.

Left flank + Kanamycin resistance + Right flank

{{{tech}}}

BBa_K1424005.

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

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