The protocols listed refer to cDNA library construction and preliminary differential screening procedures. They are mainly adaptations of standard procedures as per Sambrook et al. (1989) where theoretical background information can be found. Other publications are referred to in the text.
Nick Talbot lab /John Hamer lab protocols. Extra information available from:
N.J.Talbot@exeter.ac.uk
1. cDNA Library Construction
Based upon blast-infected rice leaf cDNA library (Talbot, Ebbole & Hamer-Plant Cell 5:1575-90, 1993). RNA extracfions were carried out by a Guanidinium Isothiocyanate procedure (Sambrook et al. 1989) and poly(A)+ purified over Oligo(dT) columns. The library was constructed in lambdaGEM4 (Promega).
(a) ANNEALING REACTION
1.Resuspend Xba-oligo(dT) primer adaptor ( or an oligo-dt primer) in sterile water, at a concentration of 1 ug / ul.
2. Combine the following on ice.
Poly(A+) RNA from healthy rice leaves 10 ul (10 ug poly(A)+RNA)3.0 M NaOAc 1.0 ul Ethanol 25 ul
Chill at -20℃, spin down RNA pellet. Dry down in a speed vac dessicator.
3. Resuspend in 10-ul of primer.
4. Heat to 65℃ for 5.0 min.
5. Quick chill in ethanol ice bath.
(b) FIRST-STRAND SYNTHESIS
(After Brown and Kafatos, 1988: J.Mol,Biol, 2O3:425-437)1. To the annealed oligo and poly(A)+ RNA add the following on ice:
1.25X RT Buffer (-CTP) 80ul 5-methyl dCTP 10ul RNasin (Promega) 1 ul M-MLV RT (BRL) 2 ul1.25X RT Buffer (-dCTP) is:
1.O M Tris-HCl (pH 8.7) 100ul 100mM 100 mM DTT 10 ul 10 mM 1.O M KCl 40 ul 40 mM 1.0 M MgCl2 8 ul 8 mM 100 mM dGTP (Pharmacia) 8 ul 0.8 mM 100 mM dATP 8 ul 100 mM dTTP 8 ul 80 mM Na4P2O7 5 ul 4.0 mM (500 ug/ml) actinomycin D 100 ul 50 ug H20 597 ul
