Nick Talbot lab /John Hamer lab protocols. Extra information available from:
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).
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 C, spin down RNA pellet. Dry down in a speed vac dessicator.
3. Resuspend in 10-ul of primer.
4. Heat to 65 C for 5.0 min.
5. Quick chill in ethanol ice bath.
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 ul
1.25X 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 Na4P207 5 ul 4.0 mM (500 ug/ml) actinomycin D 100 ul 50 ug H20 597 ul
Mix well and dispense in 100 ul aliquots and store at -70 C.
2.Incubate the reaction at 40 C for 1.0 hour.
3.Terminate reaction by placing on ice and extract with 100 ul of phenol- chloroform. Remove 95 ul of aqueous and reextract organic phase with another 100 ul of O.1 X sterile TE
4.Precipitate the DNA:RNA hybrid with 0.1 vol sodium acetate (20 ul) and two volumes (440 ul) of ethanol. Chill in an ice bucket for 10 min. and pellet by centrifugation for 20 minutes in the cold.
5.Drain ethanol and resuspend pellet in 20 ul of TE and precipitate with ammonium acetate (20 ul of ammonium acetate, 80 ul of ethanol). Allow to stand at least 2-3 hrs (O/N is better according to BRL). Spin down at room temperature. Wash pellet in 70% ethanol.
6. Dry in the Speed Vac and resuspend in 10 ul of O.lX TE .
1. To the dried cDNA pellet add the following:
1st strand cDNA 10.0 ul 0.4M Hepes pH 7.6 25.0 ul 6O mM MgCl2 10.0 ul 1.0 M DTT 1.0 ul 1.O M KCl 6.0 ul 5.O mM dNTP mix 10.0 ul 10 mM B-NAD 1.5 ul P32-dCTP 10.0 ul DNA Pol 1 15.0 ul RNase H 3.0 ul E. coli ligase 1.0 ul dH20 7.5 ul
2. Mix well on ice and incubate at 14 C for 1.0 hr. then shift to RT for an additional hour.
3. Stop the reaction by placing on ice. Perform one phenol/chloroform extraction (reextracting the organic layer) and precipitate once with sodium acetate and ethanol. Dry the pellet and resuspend in 5 ul of O.1X TE.
1OX NTB (Maniatis nick-translation buffer) 3 ul 5mM dNTPs 1 ul H20 20 ul T4 DNA polymerase 0.5 ul2.Incubate at 37 C for 20 min. Adjust aqueous volume to 100 ul (by adding 70 ul of O.1 X TE). Perform one phenol chloroform extraction, reextracting the aqueous volume and precipitate the DNA by ammonium acetate precipitation. Wash pellet with 700/0 ethanol and dry in speed vac. Resuspend cDNA in 10 ul of O.1 X TE.
1. We prepare and test linkers as in Sambrook et al. (1989).
Combine the following on ice.
double -stranded cDNA 10.0 ul 1O X Ligation buffer 2.0 ul Kinased linkers 5.0 ul H20 2.0 ul 1OmM ATP 0.5 ul T4-DNA ligase 0.5 ul
Mix well and incubate at 140C overnight. Check all centrifuges and work area for radioactivity.
1O X Restriction buffer 10.0 ul 1OX Gelatin (1 mg/ ml) 10.0 ul H2O 56.0 ul Xbal 2.0 ul
Incubate for 1.0 hr at 37 C
Add 2.0 ul of EcoRl and continue incubation for 2 more hours. During this time set-up a 5% acrylamide gel. (Note: This step for directional Eco-Xba cDNA cloning)
3. Remove restriction digest to ice and store at -20 C. Remove 3.0 ul for gel analysis. Combine with 5 ul of TE with bromphenol blue stop dye.
4. Electrophorese on a 5% acrylamide gel, CDNA samples before and after digestion with restriction enzymes. Include size standards. Dry gel and expose overnight to film.
5. Set-up Biorad agarose A-50M column. Phenol-chloroform extract cDNA to remove restriction enzymes. Reextract organic phase. Precipitate with NaOAc and ethanol. Spin drain pellet and resuspend in O.1X TE.
6.Load on to column pre-equilabrated with O.lX TE. Collect 200 ul fractions in eppendorf tubes (approximately 40). Monitor radioactivity. Assay 2.0 ul of every second fraction on a 1.0% TAE agarose gel if you have doubts. The separation of cDNA from kinased linkers should be obvious!!
7.Dry gel and expose to film. Pool fractions with CDNA equal to or greater than 500 bp (if you want). Concentrate by butanol extraction and ammonium acetate precipitate.
8.To the dried cDNA pellet add:
H20 6.0 ul 1O X ligation buffer 1.0 ul Lambdagem4 DNA* 2.0 ul 1OmM ATP 0.5 ul T4 Ligase 0.5 ul
Incubate at 10-150C overnight continue at RT the next day.
9. Packaging follows next day.
* Prepping the Lambda vector before cloning.
1. We prepare 25 ug of lambda vector. The Lambda DNA is (25ug/100 ul of O.1 X TE) heated to 68 C for 10 min. and allowed to cool slowly at room temp.
The cos-annealed Lambda DNA is then ligated in a total volume 200 ul overnight
2. The cos-ligated lambda DNA is then adjusted to 300 ILI with 10 X RE, and dH20 and digested with Eco and Xba overnight.
3.The enzymes are heat inactivated, and an appropriate amount of CIAP is added (we calculate the required amount according to the formulas given in the product sheet from Boahringer).
4.After phosphatase treatment at 37 C for 45 min. we inactivate with a 1/10 th volume of EGTA, heat at 70 C for 45 min., and do 2 phenol-chloroform extractions, and ethanol ppt the lambda DNA.
5.We repeat enzyme digestion and phosphatasing a second time. The final Lambda DNA is resuspended in 20 ul. Very low backgrounds of nonrerombinants are obtained (6 pfu/ug of vector packaged).
6. Packaging by standard procedures using Stratagene packaging extracts.
1.Plate out at library at high density using standard procedures (Sambrook et al. 1989). Allow plaques to become visible but not to large (5-6h at 37 C).
2.Chill plates for at least two hours before carrying out plaques lifts.
3.Carry out duplicate plaques lifts. First lift gets 30 sec contact with plate, second lift 2 min.
4.Incubate nitrocellulose discs in denaturation buffer 30 sec, then neutralisation buffer for 30 sec. Standard recipes.
5.Wash filters in 2 x SSPE, rubbing off any adhering agar (This doesn't affect plaque blots).
6.Air dry the filters, then bake at 80 C for two hours under vacuum (Call me a traditionalist).
7. Prehybridise o/n, Then hybridise with + and - cDNA probes.
8. First lifts always get probed with the - probe (in this example healthy rice leaf cDNA).
Second lifts get the + probe (Infected rice leaf CDNA). cDNA probes are made by carrying out a first strand cDNA reaction with 1 ug Poly(A+) RNA as per the recipe above. The reaction is incubated for 1 h. The probe is purified through a biogel p60 column and denatured with boiling or NAOH treatment before use. This is essentially standard (kits available), but simply follow the first steps of the library protocol if in doubt.
9. Hybridise 12h. You can also do a 48h hybridisation to screen for low abundance cDNAs.
10. Autoradiograph as usual.
11. Carry out secondary screens In the same way. Phage DNA can be extracted conventionally or use single plaque PCR, or PCR from a plaque pure SM -70 C stock to get the cDNA insert. Use T7/SP6 primers for GEM4, or T3(T7 for ZAP. Sequencing can be direct based on solid phase protocols, or conventional dsDNAor ssDNA protocols.
12. Southern blot your clones against genomic DNA sooner rather than later.
Any problems with the above, and I am happy to offer advice by E-mail. Much of the above can be supplied in kit forms by major suppliers, particularly:
Stratagene, Promega and BRL.
Their tech. service are normally pretty helpful.