Oneill Lab:Random Priming

From OpenWetWare

Jump to: navigation, search

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

Back to O'Neill Lab Protocols

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

Contents

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
  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

Hybridization

  1. Produce blot by Southern Transfer or other method
  2. Place fixed blot in a hybridization bottle with an appropriate amount of Church's Hyb Solution.
    • For small bottles 10 ml
    • For large bottles 20 ml
  3. Prehybridize blot in hyb solution at your hybridization temperature for two hours or longer.
  4. When random priming incubation and prehybridization are complete, transfer purified, labeled probe to a 15 or 50 ml Falcon tube.
  5. Heat tube to 100°C for 5 minutes
  6. Pour warm Church's from prehyb into tube with denatured probe.
  7. Discard any excess hyb solution
  8. Pour probe from Falcon tube into hyb bottle
  9. Cap and return to hybridization oven.
  10. Hybridize at temp. for 12-16 hours or longer.

Washing

The extent which a given hybridization experiment will need to be washed can vary greatly. You will need to monitor signal from the blot throughout the washing process. Separating real signal from background radiation can be difficult without exposing film. Ultimately, this will be the only reliable method for determining if you have washed sufficiently.

  1. Prepare wash solutions
    1. 2x SSC, 0.1% SDS
    2. 1x SSC, 0.1% SDS
    3. 0.5x SSC, 0.1% SDS
    4. 0.1x SSC, 0.1% SDS
      • Prepare about 500 ml of each, though you can create more for long term storage.
  2. After hybridization is complete, pour probe back into the falcon tube used in step 4 of #Hybridization
  3. Place blot in a suitable container, and cover with room temp or heated 2x wash solution.
  4. Rinse blot with shaking for 5 minutes to remove excess probe.
  5. Remove rinse, and cover with 2x wash solution heated to hybridization temperature
  6. Wash for 15-20 min.
  7. Remove wash, and proceed down SSC gradient, checking for signal after each wash.
  8. When washes are finished, wrap blot in plastic wrap
  9. Place in radiography film cassette
  10. In dark room, lay a piece of x-ray film over blot. Seal the cassette
  11. Place cassette in -80°C freezer for overnight exposure
    • The exposure time may need to be increased or decreased depending on the nature of the probe and blot, and the intensity of the signal after washing.
  12. The next day, allow the film cassette to thaw before developing.
Personal tools