Colony/Plaque Blotting - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Colony/Plaque Blotting

Colony Blotting
1. Grow bacteria on agar containing the appropriate selective antibiotic until colonies
are approximately 0.5-1mm in diameter.
2. Place a Hybridisation membrane cut to the size of the plate on to the surface of the
agar. Orientation points should be marked at this stage by piercing the membrane
at three asymmetric points with a sterile needle.
3. Replica filters may be prepared from the initial colony lift at this stage by pressing a
second Hybridisation membrane firmly on to the template membrane for a few
seconds. The simplest way to do this is by sandwiching the membranes between
glass plates lined with a few sheets of Whatman 3MM paper, and pressing them
firmly together. Carefully separate the membranes to avoid smearing the colonies.
This replica, and any subsequent replicas, should also be marked with orientation
points.
4. The replica membranes are then placed colony side up on to fresh agar plates
containing the appropriate selective antibiotic and incubated at 37°C until colonies
of 0.5-1mm are present on the surface of the membrane. At this stage, it is possible
to amplify low copy number plasmids (relative to cellular DNA) by transferring the
filters on to agar plates containing chloramphenicol at 200µg/ml and incubating for
10 hours at 37°C.
5. Place the Hybridisation membranes on Whatman 3MM paper saturated in
neutralising solution for 3 minutes then repeat with fresh neutralising solution.
6. Rinse the membranes briefly in 2 x SSPE (SSC), then air-dry on a sheet of filter
paper.
7. Fix the DNA to the membranes either by baking for two hours at 80°C, or by UV
crosslinking.
8. After fixing the DNA, membranes may be stored between sheets of Whatman
3MM paper in sealed plastic bags at 4°C.
9. Prior to Hybridisation, it may be advantageous to carry out a pre-washing step to
remove colony debris and hence reduce background noise.
© Thermo Scientific, May 2003. Issue 7
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