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. Pellet 500 ml ON culture (4 krpm, 10' @ 4oC).
2. Wash 1X in TE.
3. Resuspend in 8ml Lysis Buffer and transfer to 50ml cfg. tube.
4. Add 5ml 0.5M EDTA + 1.5ml 5mg/ml lysozyme, mix gently.
5. Incubate 15' on ice.
6. Add 15ml Triton Mix and mix gently.
7. Incubate 15' on ice.
8. Spin 18 krpm, 45' @ 4oC.
9. Carefully pour the supernatant into a 50ml tube. Add CsCl2 @ 100% w/v.
10. Add 3ml 10mg/ml EtBr.
11. Centrifuge 40 krpm, 20h @ 20oC. (VTi50 rotor).
12. Harvest the plasmid DNA with a 16 or 18G needle. Remove the EtBr by extracting with
salt-saturated isopropanol or butanol. Dilute the prep with 3-4 vol. TE and precipitate with
2 vol. EtOH.
TE
10mM Tris-HCl, pH 8.0
1mM EDTA
Lysis Buffer
50mM Tris-HCl, pH 8.0
10% Sucrose
0.5M EDTA
5mg/ml Lysozyme
(freshly prepared in lysis buffer)
Triton Mix
50mM Tris-HCl, pH 8.0
62.5mM EDTA
0.1% Triton X-100
Cesium Chloride
10mg/ml Ethidium Bromide
CsCl2-saturated Isopropanol or Butanol
acc 2/90
