MATERIALS AND METHODS


LB Agar

Tryptone 10 g
Yeast extract 5 g
Sodium chloride 5 g
Water To 1 litre
  • pH to 7.5 with NaOH
  • Add 15g agarose just before autoclaving.
  • Autoclave.Allow medium to cool to 50oC before adding ampicillin to a final concentration of 100 ug/ml (1/1000 dilution of a 100 mg/ml ampicillin stock solution). Plates can be stored for 1 month at 4oC or 1 week at room temperature.



Ampicillin

Ampicillin 1 g
70% Ethanol 10 mL
  • Filter sterilise and store 1ml aliquots at -20oC.




Anticoagulant (mammalian)

Trisodium citrate 3.8 g
Sodium chloride 9 g
Water To 100 mL
  • pH to 7.4




Sodium carbonate buffer

Sodium carbonate 0.318 g
Sodium hydrogen carbonate (bicarbonate) 0.586 g
Sodium azide (optional) 0.004 g
Water to 100 mL
  • pH to 9.5
  • Final concentration is 0.1M



Coomassie Blue Stain

Coomassie Blue 0.25 g
Methanol 250 mL
Acetic acid 50 mL
Water 200 mL
  • Stain for 1 hr - overnight on a rocker platform.
  • Optional: gel can be pre-fixed for 10 min with 10% acetic acid or 10% trichloroacetic acid, before staining.




Coomassie Blue destain

Methanol 500 mL
Acetic acid 100 mL
Water 400 mL
  • Wash 5 x 3 min on rocker platform.
  • For a greatercontrast, extended destain may be carried out.
  • Wash 1-2 x 3 min in Milli Q water.




Bromodeoxyuridine

Bromodeoxyuridine 3.07 mg
Water 20 mL






sham.nair@mq.edu.au
shamnair@gmail.com



Sham Nair 2014