1. Be sure to use the appropriate laboratory technique when handling radioactive and toxic materials. Consult a laboratory safety manual if you are in doubt regarding what proper practices are. Methyl mercuric hydroxide is quite toxic and should be handled with extreme care in a fume hood. This toxicity is balanced by its extremely potent and reversible denaturing activity. Each new batch of methyl mercuric hydroxide should be tested for performance in denaturing gel electrophoresis as described in ref. 9. If sharp bands are not observed, purify the methyl mercuric hydroxide by stirring for 2 h at room temperature with a mixed-bed resin, such as Amberlite MB-1 or equivalent. Remove the resin and other debris by passing through a 0.2-|im syringe filter. Store in small aliquots at -70°C. Be sure to dispose of mercury waste appropriately.
2. This step is required to repair the cDNA and render it blunt-ended prior to linker ligation.
3. This additional blunting is required to repair any damage caused by £coRI methylase. We have noticed variable amounts of nuclease activity in methylases and think this extra step is prudent.
4. We usually buy phosphorylated linkers as well as unphosphorylated ones, since it is more efficient to synthesize the linker with the phosphate on than to add it later enzymatically.
5. One should be careful that the linkers are in sufficient excess in this reaction. This, of course, depends on the yield of cDNA. Assume an average cDNA length of 1 kb in the first strand reaction and calculate the number of picomoles of ends. Be sure that the linkers are in 50- to 100-fold molar excess to ensure that cDNAs are not artifactually ligated to each other.
6. The addition of T4 RNA ligase stimulates blunt-end ligation up to 10-fold. Furthermore, RNA ligase is capable of ligating linker molecules to RNA remaining at the 5'-end of cDNA. It is quite probable that some of the longest cDNA molecules will have a few nucleotides of RNA left at the 5'-end, which RNase H can not remove. If RNA ligase is not added, then linkers can not be added to this end and these cDNAs will be lost from the library.
7. Check for ligation and digestion by running an 8% polyacrylamide gel in TBE before and after digestion samples. A typical protein gel apparatus is appropriate. Run the gel at 300-400 V, and stop it when the bromophenol blue goes half way down. Expose to film for several hours, or dry the gel down and expose for an appropriate time. Expect to see a ladder of linkers in the undigested sample, it should be gone in the £coRI-digested sample, and there should be two bands near the bottom of the gel. If the XhoI digest was done separately, expect to see one additional band larger than the two in the £coRI digest. If the ligation or either digestion did not work, then go back to the blunt-ending step, and repeat carefully checking each step individually.
8. Alternatively, the £coRI and XhoI digests can be performed together using buffer H, but this does not permit checking that each step has worked. We usually do the digestions separately when testing new batches of enzymes and reagents, but otherwise do them together to save time.
9. Use a sterile, plugged, individually wrapped plastic pipet suitable for tissue culture. Score the pipet in the middle of the cotton. Break off and remove the cotton. Make a tiny ball of polyester wool, push into the column top with forceps, and blast into the bottom with compressed air or gas (available at most lab benches). Polyester wool used for aquarium filtration and is available at most pet shops.
10. Proper technique is critical here for good separation. Allow the liquid in the column to drain to the top of the bed. Apply 50 ||L of cDNA carefully, and allow to run in. While this is going on, reattach the reservoir, and carefully add column buffer when the cDNA has fully entered the column. Of course, the column should not be allowed to run dry at any point or else resolution will be compromised.
11. Using Sepharose Cl-4B and the column system described above, the first fraction following the cDNA peak is about 500 bp. Pool the fractions from here to the beginning.
12. Take care not to overdry the DNA, or it will be quite difficult to resuspend. One to 2 min in the Speed-Vac are sufficient.
13. Stratagene recommends a 2-h incubation at room temperature. We have used up to 6 h with equally good results.
14. Although it is slightly more trouble to maintain cells on minimal plates and grow them in minimal media, the increased plating efficiency and reproducibility are worth it. Some strains, most notably C600 HflA150 and Y1090, throw off resistant mutants at a high rate. Growth on media with maltose as the carbon source minimizes this phenomenon.
15. Proteinase K works well at elevated temperatures, but is denatured at temperatures above 65°C. This is a good step to let go overnight if desired.
16. When precipitating large amounts of DNA, you will get cleaner precipitates by adding the alcohol first, mixing well, and then adding the salt and mixing well again. Mix well, and store on ice, -20°C, or -70°C for 10+ min. For reasonable amounts of DNA, these three are about equivalent. The centrifugation time is much more critical. We typically use ice for 30 min followed by a 20 min spin at room temperature. For small quantities of DNA, overnight at -20°C gives superior recoveries.
17. If the production of a subtracted cDNA library is desired, then one is practically limited to using poly (A)+ RNA from the target tissue as the source of material. If a subtracted probe is being prepared, then either poly (A)+ RNA or RNA synthesized in vitro from an existing library is adequate. Whenever there is sufficient poly (A)+ RNA available, its use is preferred to minimize changes in complexity of the target RNA.
18. There are a number of high-quality kits available for labeling DNA by random priming. If you choose a commercial kit, be sure that it can be adapted to use two radionucleotides, since the probe must be of very high-specific activity to detect rare clones.
19. The choice of nitrocellulose or nylon membranes depends on the number of times the library will be screened. For one to three screenings we prefer to use sup ported nitrocellulose (e.g., BAS/NC, Schleicher and Schuell, Keene, NH) owing to its inherent high signal-to-noise ratio. If the library may be screened three or more times, them it makes sense to use a nylon membrane such as Nytram (Schleicher and Schuell). We have has intermittent difficulties with nylon membranes from other manufacturers and recommend each batch prior to using it.
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