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Preparation of Riboprobe

Reagents:

Stratagene Transcription Kit ( Cat#20340 or 20341)

³⁵S-rUTP (NEN Cat# HEG739H 1mCi ³⁵S-UTP)

RNA polymerase and Trascription buffer (Promega)

QIAquick Nucleotide Removal Kit (QIAGEN Cat# 28304)

Use 1 ug linearized plasmid DNA as the template.

Keep all reagents on ice.

Mix Following three reagents

5X Transc buffer 6ul

0.75M DTT 1ul

10mM rACG 3ul Mix

Total 10ul

1. Mix at RT in the following order: (Standard 1X transcription 30ul)
   
   H₂O 12 ul
   Plasmid 1ul (1ug/ul)
   Three reagents 10ul
   Rnase In 1ul Mix
   Polymerase 1ul (20units/ul)
   ³⁵S-rUTP 5ul
   Mix and spin down X2
   
   
2. Incubate at 37°C for 2hours.
   
   
3. Add 2ul Rnase free Dnase to destroy DNA template. Incubate at 37°C for 15 min.
   
   
4. Use QIAquick Nucleotide Removal Kit to remove unincoporated nucleotides
   ( Add 50ul EB buffer to elute the probe )
   
   
5. Measure probe cpm: Add 2ul probe to 2ml H₂O soluble scintillation fluid.
   
   
6. The count of 2ul probe usually are around 1-2million cpm.

 

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