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Protocol for Radioactive RiboRuler™ RNA Ladder Labeling using T4 Polynucleotide Kinase

Note

The ready-to-use versions of RiboRuler™ RNA ladders cannot be radiolabeled with T4 Polynucleotide Kinase.

I. Dephosphorylation. RNA ladders are made of 5'-phosphorylated transcripts. Therefore, the following dephosphorylation step is needed.

1. Prepare the following reaction mixture:

   RiboRuler™ Low Range RNA Ladder (50 pmol of 5'-termini) or RiboRuler™ High Range RNA Ladder (16 pmol of 5'-termini)	8 µl
   RiboLock™ RNase Inhibitor	1 µl (20 u)
   10X reaction buffer for CIAP (supplied with Calf Intestine Alkaline Phosphatase (CIAP))	2 µl
   DEPC-treated Water	to 18 µl
   Calf Intestine Alkaline Phosphatase (CIAP)	2 µl (2 u)

2. Mix well and incubate at 37°C for 30 minutes.
3. Extract proteins from the mixture with a 20 µl aliquot of Tris-saturated (pH 8.0) phenol and chloroform mixture. Save the upper aqueous phase and extract it twice with 20 µl of chloroform.
4. Precipitate the RNA by adding the following: 1 µl of 3 M sodium acetate (or 0.5 µl of 2 M sodium chloride), 55 µl of 96% ethanol (or 20 µl of isopropanol) and then centrifuge the mixture for 20 min at 10,000 rpm in a microcentrifuge.
5. Discard the supernatant and dissolve the air-dried pellet in 10 µl of DEPC-treated Water.

II. Labeling

1. Prepare the following reaction mixture:

   RiboRuler™ Low Range RNA Ladder or RiboRuler™ High Range RNA Ladder	1 µl 2.5 µl
   [gama-32P]-ATP (5000 Ci/mmol, 10 µCi/µl)	5 µl (10 pmol)
   RiboLock™ RNase Inhibitor	0.5 µl (10 u)
   10X buffer A for forward reaction (supplied with T4 Polynucleotide Kinase)	1 µl
   DEPC-treated Water	to 9 µl
   T4 Polynucleotide Kinase	1 µl (10 u)

2. Mix well and incubate at 37°C for 30 minutes.
3. Stop the reaction by adding 1 µl of 0.5 M EDTA (pH 8.0) and extract the mixture with an equal volume (11 µl) of chloroform.
4. Determine the efficiency of label incorporation.
5. Load the ladder on the gel.

Note

• ATP concentration should be at least 1 µM. If [gama-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the total ATP concentration can be brought to 1 µM with non-labeled ATP.

Ordering Information

RiboRuler™ Low Range RNA Ladder RiboRuler™ High Range RNA Ladder T4 Polynucleotide Kinase RiboLock™ RNase Inhibitor Calf Intestine Alkaline Phosphatase (CIAP) 0.5 M EDTA, pH 8.0 ATP DEPC-treated water

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Updated vasario 01, 2008 09:51