Home  Contacts  Order  Catalog  Support  Search  Alphabetical Index  Numerical Index  Restriction Enzymes  Modifying Enzymes  PCR, qPCR, RT-PCR & dNTPs  Molecular Cloning  Nucleic Acid Purification  Molecular Labeling & Detection  In vitro Transcription  Electrophoresis Products  Nucleotides  Transfection Reagents  Reagents S U P P O R T

Purification of DNA from Cultured Eukaryotic Cells
(with Proteinase K)

Determine the number of cells. The following protocol is used for 1-2 x 10⁶ cels.

1. Centrifuge for 5 min at 3000 rpm in a microcentrifuge to collect the cells.
2. Wash the pellet twice with PBS (137 mM NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 2 mM K₂HPO₄, pH 7.4).
3. Resuspend the pellet in 0.5 ml of lysis buffer (10 mM Tris-HCl (pH 8.5), 5 mM EDTA, 200 mM NaCl, 0.2% SDS). Incubate at 60°C for 5 minutes.
4. Add 2.5 µl of Proteinase K and 5 µl of RNase A/T1 Mix, mix. Incubate at 60°C for 1 hour.
5. Add 250 µl of 5 M NaCl, mix incubate on ice for 5 min to precipitate protein.
6. Centrifuge for 15 min at 10,000 rpm in a microcentrifuge.
7. Transfer supernatant to a fresh tube. Add a equal volume of isopropanol and mix to precipitate the DNA.
   Optional. Incubate at -20°C for up to 60 min to increase the yield of DNA.
8. Centrifuge for 10 min at 10,000 rpm in a microcentrifuge.
9. Discard the supernatant and wash the pellet with 1.2 ml 70% cold ethanol.
10. Air-dry the pellet*. Dissolve the pellet in water, nuclease-free or TE buffer.

Note

* Do not use a vacuum dryer or let the pellet dry completely. Dried genomic DNA has low solubility.
The typical yield of DNA from 10⁶ cells is 7-15 µg.

Ordering Information

Proteinase K RNase A/T1 Mix Water, nuclease-free

catalog@fermentas.com

Updated balandžio 02, 2008 08:12