Herskowitz Lab Protocol
From : JMG/RKT 7/96

Make 25% glutaraldehyde [Kodak #8648]

1. Dilute from stock
2. Mix 600ml with 4 teaspoons activated Norite Ex charcoal
3. Stir freq 5'
4. Filter through large Buchner funnel, Whatman D1 11.0cm filter paper
5. Repeat above three with 2 teaspoons charcoal & clean filters
6. Refilter into brown bottle, store at 4°C

Protocol for 1 gel

1. Gel into 200ml of 5% MeOH 10% HOAc for 1hr
2. Wash several times with nanopure H2O; preferrably leave o/n; wash at least until destain smell gone
3. 5% glutaraldehyde reduction for 30' (20ml of 25% made to 100ml)
4. Rinse several times with nanopure H2O;until all traces gone
5. Stain with for 10'
   
   21.0ml 0.36% NaOH [=1.8g/500ml]
   1.4ml NH4OH (58% is reagent ...)
   4.0ml 19.8% AgNO3 [=20g/100ml]
   --> to 200m
   
6. rinse 2X nanopure H2O; let sit in third rinse 10'
7. develop with :
   
   2ml 1.0% citric acid
   0.25ml formaldehyde (38%)
   to 500ml

Important Tips

• Use glassware not plastic - especially the tray for the gel must be glass
• Always wear gloves
• Shake gently
• Do excessive washes with water to remove fixatives

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