Phenol extraction of PBCV
Phenol extraction of PBCV (2)
Reagents
Liquid (88%) phenol, best quality by adding water to crystalline phenol
0.5M DTT solution (77mg/ml)(reduces S-S bonds)
10M LiCl (dissociations basic proteins from nucleic acids)
10% w/v SDS (denatures proteins)
1% Ponceau S (marks aqueous phase)
1% w/w Ficoll 400 (carrier)
Acetone
Procedure
To 100ul of PBCV in a 0.5ml conical plastic centrifuge tube
Add phenol (170ul)
Add DTT (~10ul)
Add 10%SDS (~10ul)
Add 10M LiCl (~5ul)
Add Ponceau S (~5ul)
Mix thoroughly. Heat 15 min at 50C, then cool
Centrifuge 1 min (3K), transfer lower phase to 1.5ml microfuge tube
Add 1ml of acetone and then 5ul of 1% Ficoll
Mix, store 15min at -20C, centrifuge 1min (6K)
Discard supernatant, drain tube, wash ppt with Acetone (ppt sticks
********** to tube), dry. Blow dry with hair drier
Dissolve pellet in cracking buffer (~50ul).
Run Gel
Gel apparatus
Running buffer (800ml) dilute from 10x, add 8ml 10% SDS
4-20% gel (in cold rm)
Standards (prestained) - 2ul
Dissolve in 75 ul cracking buffer - heat 2min at 100C
Add O.25M IAm (47mg/ml)) - 1/20 vol, 1min at 100C
Run (150V) until stain front reaches bottom
Remove gel and put in destaining solution (50C bath)
After 30' add fresh detain and Coomassie Blue