( ings. In particular UV-crosslink labe...)Tools for Detecting MSH2 Expression in Chinese Hamster Ovary Cells

ings. In particular, UV-crosslink, label a probe, prehybridize, and hybridize the probe to the blot in less than 1.5 hours. For even greater convenience and consistent results in hybridization, use the QuikHyb solution in conjunction with the PersonalHyb oven. Altogether, Stratagene makes available an integrated group of products that simplifies Northern blot analysis and leads to quick and reliable results.

Methods

Isolation, Electrophoresis, and Blotting of Total RNA: Total RNA was isolated from 5 x 10⁶ CHO cells using the StrataPrep total RNA miniprep kit.³ Duplicate samples containing 2, 1, 0.5, 0.2, and 0.1 mg of this RNA were electrophoresed on a 1.2% formaldehyde-agarose gel using Stratagenes Joule Box minigel electrophoresis system. The gel was blotted to Stratagenes Duralon-UV membrane⁴ in 10X SSC overnight. The membrane was air-dried and UV-crosslinked in the Stratalinker unit.⁴

Probe Labeling: Total RNA from CHO cells was used with the ProSTAR first strand RT-PCR kit⁵ to synthesize cDNA. An MSH2 fragment was generated from this cDNA by PCR using Taq2000 DNA polymerase and Stratagenes MSH2 primer set for RT-PCR⁶ in duplicate reactions, according to instructions. The expected single 0.9-kb band was generated. One of the reactions was purified using the StrataPrep PCR purification kit, and the other was purified with the StrataPrep DNA gel extraction kit.⁷ Each of these fragments was labeled with a-³²P-dCTP in a 5-minute reaction using Stratagenes Prime-It II random primer labeling kit.⁸ The probes were purified to remove unincorporated nucleotides using Stratagenes NucTrap probe purification columns.⁹ The specific activity of each probe was greater than 10⁹ cpm/g.

Hybridization of the Probes to Northern Blot
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