20 Expression Cloning with Xenopus Oocytes
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Materials
• Semiautomatic injector (e.g. Inject+Matic System. J.A. Gabay, Switzer- Equipment
land or Nano-ject microinjector, Drummond, USA)
• Luria Broth plates (LB), 9 cm
• Nitrocellulose or nylon fllters (Schleicher and Schuell)
• Sephadex G-50 Quick Spin columns, RNase-free (Boehringer Mannheim
or self made)
• 35 Sol-, 20-40 )lCi/ml
• K2S04
• 100 mM NaCl
• 100 mM choline chloride
• 10% SDS
• Scintillation fluid ( e.g. Emulsifier Safe, Kontron)
• Ampicillin 100 )lg/ml
• ammonium acetate 5 M
• ethanol
• 1% agarose formaldehyde RNA
Reagents
• ethyl m-aminobenzoate solution (1 g/liter in water)
Solutions
• ORII solution: 82.5 mM NaCl, 2 mM KCl, 1 mM MgC12> 10 mM Hepes
adjusted with Tris to pH 7.5
• modified Barth's solution (MBS): 88 mM NaCl, 1 mM KCl, 0.82 mM
MgS04, 0.41 mM CaCh, 0.33 mM Ca(N03)2, 2.4 mM NaHC03, 10 mM
Hepes/Tris (pH 7.4), gentamiein sulfate 20 mg/1
• Solution A: 100 mM Choline chloride, 2 mM KCl, 1 mM CaCh, 1 mM
MgCh, 10 mM Hepes/Tris (pH 7.5)
• Transcription buffer 1 x: 40 mM Tris-HCl (pH 7.9), 2 mM spermidine
and 6 mM MgCh; supplied with polymerase (Promega)
• Transcription mixture (final concentrations): transcription buffer 1 x;
0.5 mM ATP; 0.5 mM CTP; 0.5 mM UTP; 0.5 mM m7G(S')ppp(S')G (RNA
cap analog; New England Biolabs); 0.1 mM GTP; 10 mM DTT; 50 units