Team:HSHL/Experiments

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Protocols & Experiments

In the following you will find a description of our experiments, and protocols used in our iGEM project. You want to repeat our experiments and need further information? Contact us, we are glad to help you.

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Inactivationtemperatures

Temp. [°C] Time [min]
T4 DNA Ligase (NEB) 65 10
EcoRI (NEB) 65 15
PstI (NEB) 80 20
Shrimp Alkaline Phospatase (NEB) 65 5
HindIII (NEB) 80 20
EcoRII (Thermofisher Scientific) 80 20
SmaI (NEB) 65 20

We used 3 heat baths: 1 with 80°, 1 with 65°, 1 with 37°

For heat shock: increased temperature of the 37° bath to 42°

  1. RIM-Medium for plant cultivating
    2.
    1. „Murashige” + Skoog Medium including Vitamins and MES Buffer (concentration: 4.9 g/L)
    2. Adding Saccharose
      2 % (w/v) 2 g/100 mL = 20 g/L
    3. Adding Agar Type M
      0.7 – 0.8 % (w/v) 0.7 – 0.8 g/100 mL = 7 – 8 g/L
    4. Adjust pH of the medium with help of KOH (Kaliumhydroxide)
      pH 5.7 – 5.8
    5. Autoclaving of this liquid for 10 minutes for sterilizing
    6. Cool down of autoclaved medium (ca. 50 – 55 °C)
    7. Preparing IES stock solution
      1mg IES / 1 mL Ethanol scale and then storing at 4 °C
    8. For 500 mL medium 500 µl IES Stocklösung sterile in medium pipette
      for 1 L medium put 1 mL stock solution in medium
  2. SOC-Medium
    3.
    1. We used NEB SOC-Medium and prepared it according to manufacturer’s instructions
  3. LB-Medium (1L liquid)
    4.
    1. 10 g tryptone
    2. 10 g NaCl
    3. 5 g yeat extract
    4. Water
    5. (in case we needed chloramphenicol-medium, we used 50 µl for 50 ml medium)
  4. LB-Medium (solid, 1L = 50 dishes)
    5.
    1. 15 g agar agar
    2. 10 g tryptone
    3. 10 g NaCl
    4. 5 g yeat extract
    5. Water
    6. (in case we needed chloramphenicol-medium, we used 50 µl for 50 ml medium)
  5. Gel-electrophorasis protocol
    6.
    1. Pour 1x TAE buffer in electrophoresis chamber, until the agarosegel is covered by approximately 5mm.
    2. Remove agarosegel pocket spacer
    3. Add 12 µl of marker into a pocket
    4. Close electrophoresis chamber and connect to power supply.
    5. Set power supply to 100V, after 5 minutes set voltage to 200V. Run electrophoresis until the marker is approximately 1cm from the bottom of the gel (approximately 30 min)
    6. Swich off power supply, remove gel and stain it with staining solution (Sybr Safe). Afterwards wash gel and analyse bars.
  6. Agarose
    7.
    • Agarose
    • TAE 50x - Buffer
    • ddH20
    1. Prepare 30 mL of 2% Agarose solution. To do so calculate Agarose concentration by (w/v) and weight the Agarose.
    2. Add 7 mL of 50x TAE buffer into a new flask and fill with ddH2O to 350mL.
    3. Seal flask with Parafilm and mix. TAE buffer is now 1x TAE buffer.
    4. Place gel pad into electrophoresis chamber and place and add gel barriers into chamber. Afterwards place pocket spacer into gel pad.
    5. Add 30mL of 1x TAE buffer to Agarose and mix.
    6. Heat Agarose & 1x TAE buffer solution in microwave until solution is clear
    7. Pour Solution into electrophoresis chamber consistently
    8. Let gel cool for 20 min
  7. Transformation via heat shock
    8.
    1. Thaw 200 μL chemocompetent E. Coli cells on ice
    2. Add 0.5-5 μL plasmid to 200 μL chemocompetent ells
    3. Store cells on ice for 10-30 min
    4. Heatshock for 90 s at 42°C
    5. Transfer transformation reaction to 1mL SOC-Medium and incubate 1 h at 37°C
    6. Centrifuge 30 s at 11000 rpm and plate on selective LB-Medium
    7. Incubate overnight at 37°C
  8. Cloning Strategy with restriction enzymes
    9.
    • X μl 600ng Vektor
    • 3 μl 10x buffer (NEB2.1)
    • 1 μl 1. restriction enzyme
    • 1 μl 2. restriction enzyme
    • Add 30 μl dd H2O
    1. Digestion: Vector/Backbone
      Incubation 1 h, 37°C
      Inactivation 10 min, 37°C
    • X μl 100ng Insert
    • 3 μl 10x buffer
    • 1 μl 1. restriction enzyme
    • 1 μl 2. restriction enzyme
    • Add 30 μl dd H2O
    1. Digestion: Insert
      Incubation 1 h, 37°C
      Inactivation 10 min, 37°C
    1. Dephosphorylation of Vector/Backbone
      add 2 μL of Shrimp Alkaline Phosphatase (Jena Bioscience)
      Incubation 30 min, 37 °C
      Inactivation 5 min, 75 °C
    2. Phosphorylation of Insert
      add 3 μL 10x T4 Ligase Buffer (Thermo Fisher) -> contains right amount of ATP
      add 1 μL PNK (NEB)
      Incubation 30 min, 37 °C
      Inactivation 20 min, 65 °C
    3. Purification of Vector and Insert
      Any gicen Gel an PCR Purification Kit
      We recommend purification after gel electrophoresis

This wiki is designed by HSHL within the iGEM 2018 template.
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