1. A primer consisting of ~15 Ts followed by at least an XhoI site and preferably several other rare sites. The one we use is 5'(ACTAGTGCGGCCGCCTAG GCCTCGAGTTTTTTTTTTTTTTT)3'.
This has the following restriction sites (in 5' -> 3' order): SpeI, NotI, EagI, SfiI, AvrII, StuI, XhoI.
2. EcoRI linkers—octamer (GGAATTCC) (Pharmacia 5'-OH, #27-7726-01, 5'-PO4 #27-7428-01).
3. 5-methyl-dCTP (Pharmacia #27-4225-01). Store as a 100-mM stock.
4. dNTPs (Pharmacia #27-2035-01). 100 mM solutions of each.
5. 10 mM ATP (Pharmacia #27-2056-01) (dilute from 100 mM stock).
6. 0.1 M CH3HgOH (ALFA products #89691).
7. 5.8 M 2-mercaptoethanol (BME; Sigma M6250 [St. Louis, MO]).
8. 100% Ethanol (Rossvile Gold Shield or equivalent).
9. a[32P]-dATP 800 Ci/mM (New England Nuclear, Wilmington, DE, #NEG-012A).
10. y[32P]ATP 6000 Ci/mM (New England Nuclear #NEG-002Z).
12. Agarose (Pharmacia #17-0554-02 or Bio-Rad, Hercules, CA, #162-0126).
13. 40% acrylamide (19:1 acrylamide:bisacrylamide) acrylamide (Bio-Rad #1610101), bis-acrylamide (Bio-Rad #161-0201).
14. ß-NAD (BMB #775-7). The stock solution is 0.045 M in H2O.
15. Sepharose Cl-4B (Pharmacia #17-0150-01) equilibrated in column buffer.
19. Ultrapure, recrystallized phenol (BMB #100-300).
21. Sodium dodecyl sulfate (SDS) (BMB #100-155).
22. Guanidine HCl (optional) (BMB #100-173 or BRL #5502UA).
23. Guanidine thiocyanate (BMB #100-175 or BRL #5535UA).
24. Ultrapure urea (BMB #100-164).
26. Sephadex G-50 spun columns, equilibrated in TE Sephadex G-50 medium (Pharmacia #17-0043-01), or Sephadex G-50 spun columns (Pharmacia #17-0855-01).
27. LB media and plates.
28. Minimal media and plates (use maltose as carbon source).
30. All other chemicals and reagents should be at least ACS-reagent grade. You cannot go wrong by buying small quantities of ultrapure chemicals (e.g., from Aldrich) and reserving them for making cDNA buffers and solutions.
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