Team:NTNU Trondheim/Protocols

From 2014.igem.org

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<h3 class="centered">Transformation (Synechocystis sp. PCC 6803)</h3>
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<p> The transformation procedure for transforming Synechocystis can be found at the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/synechocystis-transformation-r-hill-method">PhotoSynLab wiki</a>.
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Revision as of 01:10, 16 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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Media
Techniques
Plasmids
Organisms
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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.

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

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.

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.

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.

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