Processing Q-Bot Membranes
|Boiling waterbath||95 Â°C|
|Denaturing Solution||0.5M NaOH, 1.5M NaCl|
|Neutralizing Solution||1M Tris-Cl pH 7.4, 1.5M NaCl|
|PROPK Solution||50mM Tris-Cl pH8.5, 50mM EDTA, 100mM NaCl, 1% Na-lauroyl-sarcosine|
|Proteinase K||500mg/9ml, use 3ml per 600ml PROPK|
Observe membrane and note any abnormalities about growth after spotting. Check pencil numbers and robot puncture marks.
Handle membrane with 2 forceps holding it for diagonal corners.
Place on Whatman pre-wetted in Denaturing Solution. Leave for 4 minutes.
Place membrane on fresh Whatman pre-wetted in Denaturing Solution and transfer to glass plate sitting on empty pipette tip box in boiling waterbath. Leave for 4 minutes with covered lid.
Place membrane on Whatman pre-wetted in Neutratlizing Solution. Leave for 4 minutes.
Place membrane on dry Whatman. Leave for 1 minute.
Submerge membrane under a 600ml pre-warmed (37 Â°C) PROPK Solution containing ~150mg/ml fresh Proteinase K. Do NOT shake. Leave in incubator for 30-50 minutes.
Place membrane on dry Whatman.
Cover with a NUNC. Leave overnight.
Store between dry Whatmans.