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.
(2) Differential cDNA Screen
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.
1. Bachem, C. W. B., Oomen, R. J. F. J.and Visser, R. G. F. Transcript imaging with cDNA-AFLP: A step-by-step protocol. Plant Mol. Biol. Rep. 1998; 16:157-173.
2.Bachem, C. W. B., van der Hoeven, R. S., de Bruijn, S. M., Vreugdenhil, D., Zabeau, M.and Visser, R. Visualisation of differential gene expression using a novel method of RNA finger-printing based on AFLP: Analysis of gene expression during potato tuber development. Plant J. 1996; 9:745-753.
3.Bachem, C. W. B., van der Hoeven, R. S., Lucker, J., Oomen, R. J. F. J., Casarini, E., Jacobsen, E.and Visser, R. G. F. Functional genomic analysis of potato tuber life-cycle. Potato Res. 2000; 43:297-312.
[1] Poly-A+ RNA isolation is not required for the procedure, especially when isolating RNA from small amounts of tissue. However, when total RNA is used for template preparation, DNaseI treatment is required.
[2] Since fragment isolation from PAGE can give rise to various artefacts, it is important to verify band identity using the appropriate protocols.
