Amputation andRNA Isolation

RNA Isolation

• Axolotl: 0.01% Bencocain depends of the size!!
• Wait ca. 10min before cutting!
• Collect the tissue in ca 0,5 ml fluid nitrogen and put into -80°

Homogenisation of the tissue to precipitate RNA:

1. Homogenize tissue in 1ml TRIzol per 50-100mg of tissue (using power homogenizer)

# Washing the device in 3 steps of water # devide the suspension of 1ml in 1,5 ml tubes # Add 200µl of Chlorophorm  Shake the tube and incubate it for 5’ # Centrifuge the samples with full speed for 15’ at 4°C # Transfer the aqueous phase to a fresh tube  Centrifuge it again for 5’ # Transfer it in a fresh tube precipitate the RNA by mixing 500µl Isopropanol and incubate samples for 10’  centrifuge it for 10’ at 4°C # Remove the supernatant # Add 1ml 75% Ethanol  mixing it by vortexing and centrifuge for 5’ at 4° # Dry the pellet and redissolved it with 50µl H2O and store it at -80°C

• Measure the concentration NanoDrop!!
• DNAse treatment to eliminate the contamination?