Gene Manipulation Set (part 1)


'Alexander Popper Alexander Popper / 'Katrin Stockhammer Katrin Stockhammer

GENE TRANSFER IN PLANTS

TRANSFORMATION

• Thaw out 100 ml of competent bacteria (Escherichia coli) on ice and dilute with the plasmid DNA.


• After 20 min., leave the bacteria for 90 sec. at 42°C (heat shock) and transfer again for 2–3 min. onto ice.


• Add 900 ml SOB-Medium to the preparation and shake for 1 hour at 37°C


• Plate 100–250 ml of this onto LBamp agar

AMPLIFICATION OF PLASMID DNA

• Inoculate 2–3 ml of selective medium with transgenic bacteria and shake over night at 37°C.


• Separate by centrifuging at 4000 rpm


• Remove the supernatant and resuspend the pellet in 300 ml STELI (Sucrose-Tris EDTA TritonX-100® Lysozyme).


• After 3 min. on ice, transfer to 95°C for 3 min and then separate by centrifuging for 15 min at 14000 rpm.


• Remove the pellet carefully with a sterile tooth pick and incubate the remaining supernatant containing the plasmid DNA with 1 mg RNase A for 15 min., at room temperature.


• Precipitate the DNA with isopropanol and separate by centrifuging for 15 min.


• Dissolve the washed and dried pellet in 20ul 1xTE (Tris EDTA).

CHARACTERIZATION OF THE PLASMID DNA

• Bring agarose to the boil in 0.5x TPE (Tris Phosphate EDTA) and after cooling, dilute with 5 ml ethidium bromide 100 ml.


• Ethidium bromide intercalates in the DNA and makes DNA visible through the absorption of UV rays.


• Pour the gel which is still in a liquid state onto a tray and after hardening, transfer to a buffer chamber filled with 0.5x TPE.


• Dilute the DNA specimen to be analyzed with the loading buffer and pipet into the slots.


• Separate the DNA electrophoretically at 1–5 eV/cm, according to size

TRANSFORMATION OF EMBRYONIC PLANT CELLS WITH THE HETEROLOCOUS DNA

REGENERATION OF COMPLETE TRANSGENIC PLANTS FROM THE TRANSFORMED TISSUE CULTURE

• SIM:
  IPA (isopentenyladenosine) 4.0 mg l
  NAA (Naphtol acetic acid) 0.2 mg l
  NIS (Murashige & Skoog) salts 4.4 g/l
  Sucrose 30 g/l
  Vitamix 2 ml/l
  Agar 9 g/l pH 5.8 (KOH)


• Let the regenerated shoots form, then incubate briefly in RIM (root inducing medium)


• RIM:
  IAA (indole acetic acid) 1 mg/l
  IBA (indole 3 butyrate) 0.2 mg/l
  Kinetine 0.2 mg/1


• Apply the shoot to the MS medium. Wait for the roots to form.
  MS Medium:
  MS salts 4.4 g/l
  Sucrose 30 g/l
  Vitamix 2 ml/l
  Agar 9 g/1
  pH 5.8 (KOH)