John M. S. Bartlett.pdf - Bio-Nica.info

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by most, but not all M13 polylinker enzymes (>75% digestion in 2 h by EcoRI, KpnI,
AvaI, XmaI, PstI, BamHI, SacI, and XbaI, but 7.5 (nucleic-acidgrade)
and chloroform (AR-grade). Store at 4°C in a dark glass bottle.
2. Chloroform/isoamyl alcohol (241): Store at 4°C.
3. Spin filtration device, such as Amicon Microcon (Beverly, MA) or Promega Wizard
PCR purification unit (Madison, WI).
4. Variable-speed microcentrifuge.
5. TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
6. T4 DNA polymerase.
7. 10× T4 DNA polymerase buffer: 500 mM NaCl, 100 mM Tris-HCl, pH 7.9, 100 mM
MgCl 2 , and 10 mM DTT, 500 µg/mL BSA.
8. T4 polynucleotide kinase.
9. 10× polynucleotide kinase (PNK) forward reaction buffer: 500 mM Tris-HCl, pH 7.5, 100
mM MgCl 2 , 50 mM DTT, 500 µg/mL BSA.
10. T4 DNA ligase.
11. 10× T4 DNA ligase buffer: 500 mM Tris-HCl, pH 7.8, 100 mM MgCl 2 , 100 mM DTT,
250 µg/mL BSA.
12. Calf intestinal alkaline phosphatase (CIP).
13. 10× CIP buffer: 200 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 10 mM ZnCl 2 .
14. Restriction enzymes at 10 U/µL and 10× reaction buffers.
15. 0.5 M EDTA, pH 8.0.
16. 3 M sodium acetate, pH 5.3.
17. Absolute ethanol, stored at –20°C.
18. Ultrapure stock of all four dNTPs: Make 20 mM stock in sterile water and store at –20°C.
Avoid freeze/thaw.
19. 10 mM ATP: Store in aliquots at –20°C. Avoid freeze/thaw.
20. 60% (w/v) PEG 8000 in water, filter-sterilized. Store at room temperature away from
direct sunlight.
21. Ultrapure low-melting-point agarose.
22. Glass bead DNA isolation kit, such as Geneclean (Bio101, Madison, WI).