Hydrate 5 g Sephadex G-50 beads (Sigma Aldrich) in 100 ml deionized H2O. DEPC-treat for 30 min. and autoclave. Store incomplete stock at room temperature. Before use, add the following RNase-free solutions:
0.5 ml 0.2 M EDTA
1 ml 1 M Tris pH 8.0
0.5 ml 20% SDS
Store complete G-50 solution at 4° C.
Remove and discard the plunger from a 3 ml syringe (BD Biosciences) and place the barrel of the syringe into a 15 ml conical tube (Corning). Plug the syringe with a small amount of glass wool (a plug about half the size of a penny).
Swirl complete G-50 solution to resuspend beads.
Add 2 ml G-50 solution to the empty column.
Spin for 1 minute at 1,000 x g in benchtop centrifuge.
Add 200 μl DEPC-treated deionized H2O to each column. Spin.
Repeat wash twice more for a total of three washes.
Remove syringe barrel to a fresh 15 ml conical tube.
Collagenase solution
75 mg collagenase from Clostridium histolyticum (Sigma Aldrich)
25 ml 0.1 M KPO3+ (pH 7.4)
MBSH buffer
88 mM NaCl
1 mM KCl
2.4 mM NaHCO3
0.82 mM MgSO4 X 7H2O
0.33 mM Ca(NO3)2 X 4H2O
0.41 mM CaCl2 X 6H2O
10 mM HEPES (pH 7.6)
Oocyte Culture Medium
50% L15 medium
15 mM HEPES (pH 7.6)
1 mg/ml insulin
100 mg/ml gentamicin
50 U/ml nystatin
50 U/ml penicillin
50 mg/ml streptomycin
MEMFA solution
0.1 M MOPS (pH 7.4)
2 mM EGTA
1 mM MgSO4
3.7% formaldehyde
Computing RNA yield
Determine CPM in “input” and “incorporated” samples using a standard scintillation counter.
incorporation = (“incorporated”) / (10 x “input”)
Typical incorporation values range between ~0.03 and 0.10.
Maximum theoretical yields for different polymerases:
T7, T3, SP6 – 2.64 μg
Reaction yield in μg = (maximum yield of polymerase used) X (incorporation)
Dilute RNA to 50 nM = (μg RNA) / 320 / (length of RNA in bases) / (5X10-8)
The reaction usually yields ~50-100 μl of RNA at 50 nM.