Secondary Filter Preparation

• You will need a very large area of desk space cleared off before beginning

• Cut off about a 3 feet piece of plastic wrap and lay flat on the desk.

• Cut a piece of filter paper about 24 cm wide X 48 cm long

• Place the filter paper on top of the plastic wrap and roll the edges up to fit the size of the filter paper. This will form a barrier to keep the buffer in the paper.

• Repeat this five more times to make a total of six filter papers and line them up juxtapose.

• Now soak the paper with the respective buffer listed below ~100ml

Buffer order Reagent time

1 10% SDS 5min

2 0.5M NaOH/ 1.5M NaCl 5min

3 0.5M Tris (pH 8.0)/ 1.5M NaCl 5min

4 0.5M Tris (pH 8.0)/ 1.5M NaCl (repeat) 5min

5 2x SSC/0.1% SDS 5min

6 2X SSC 5min

• After pouring the respective buffers, take a pipet and roll the bubbles out from underneath the paper and make sure the entire paper is soaked.

• Take out the filters from the 37°C incubator from overnight.

• Using filter forceps, grab the diagonal corners of the filter and lift off of the agar and place on the soaked filter paper starting with the SDS buffer first.

• It is best place the sagging point of the filter on the buffer first and roll the rest of the filter out on to the paper with buffer to try and avoid trapping bubbles under the filter. You want to avoid bubbles as this will result in improper preparation in the area.

• The size of the paper allows for two filters to be processed on the same buffer at a time.

• Each filter should be placed on each buffer for 5 minutes and then picked up with the filter forceps and moved to the next consecutive paper with buffer in the same manner described above.

• Then the next two filters can be placed on the first filter with SDS and moved along every five minutes on the buffers behind the first two.

• After the filter has spent five minutes on the final buffer of 2X SSC, place on a dry piece of filter paper and let dry for a couple of hours.

• After drying place the filter into a crosslinking oven at 120,000 microjoules per cm².

• Now the filters are ready to be cut up into the six fields and prehybridized.

* Be careful about handling the filters that you do not get fingerprints on and always handle with gloves and filter forceps when possible.