Oneill Lab:Random Priming

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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This protocol is adapted from Sambrook's Molecular Cloning 3rd Edition.

Preparing DNA template

• PCR amplify your probe sequence. Probe length should be around 500 bases.
• Gel purify the probe band from a [NuSieve] TAE gel. Digest overnight with β-agarase enzyme.
• Estimate DNA concentration by running a small portion of purified probe on a test gel.

Random Priming Setup

1. Combine 25ng template DNA with water to a final volume of 29 μL
2. Add 125ng (1 μL of 125ng/μL solution) of random primers
3. Heat mixture to 100°C for 5 minutes
4. Place immediately on ice for a 1 minute
5. Quick spin contents to bottom of tube, then return to ice
6. Add the random priming reagents
   • 5x random priming buffer - 10μL
   • 1mM dGAT Mix - 5 μL
   • 5 units Klenow Fragment - 1 μL
   • [α-³²P]dCTP - 5 μL
7. Incubate at room temp for 1 hour

Clean-up

Purify labeled probe on GE illustra™ ProbeQuant G-50 Micro Column

Column Preparation

1. Resuspend resin by vortexting
2. Loosen cap by 1/4 turn and snap off bottom closure
3. Place column in microcentrifuge tube
4. Spin column for 1 minute at 735xg (3000 rpm)

Sample Application

1. Place column in a new microcentrifuge tube
2. Slowly apply sample to column without disturbing resin bed
3. Spin column at 735xg for 2 minutes
4. Dispose of column properly