Miscellaneous Reagents and Supplies for cDNA Synthesis

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.

Was this article helpful?

0 0

Post a comment