Welcome to the Home of Vesicle Trafficking
A small repository of synaptic protein information
Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml).
Caution! In converting these protocols to HTML, some units have been scrambled.
1. Dissect tissue asceptically. Tease into a single cell suspension in 20ml TEN using the
rubber end of a syringe plunger and a metal sieve (60 micron works well).
2. Add RNase A to 100mg/ml and incubate 10' @ RT.
3. Add 1ml 20% SDS and incubate an additional 10' @ RT.
4. Add 1ml 10mg/ml Proteinase K and incubate 3h @ 50oC.
5. Gently phenol extract 10'. Spin 10' in a table top centrifuge.
6. Harvest the aqueous phase with a wide-bore transfer pipet and extract with phenol- chloroform. Spin 10'. Repeat extraction if necessary.
7. Harvest the aqueous phase. Dialyze ON against 4 changes of TE.
TEN 50mM Tris-HCl, pH 8.0
100mM EDTA
200mM NaCl
RNase A: 20mg/ml in H2O, boil 10' and allow it to cool to RT; store @ -20oC.
20% SDS (w/v)
Proteinase K: 10mg/ml in ddH2O prepared fresh or from frozen stock.
TE 10mM Tris-HCl, pH 8.0
1mM EDTA
acc 1/90
