á
Turn on hybridization
oven (Fisher isotemp 13 247 31) and heat to 58¡C
á
Label the appropriate
number of 300mm hybridization tubes (no more than 7 filters per tube)
á
Add 75ml Church buffer
(see recipies) to each tube and place in the hybridization oven for at least 30
minutes
á
Heat 1L of church buffer
to about 60¡C
and place the filters facing down into a Clear
Magazine Snap Case (14Óx 8 5/8Óx 3 7/8Ó The Container Store 584070). Pour the church buffer over the filters
into the container. Flip the
filters individually in order to ensure the filters are not sticking together and
will hybridize properly. They now
should have the labels and DNA facing up.
á
Roll the filters up with
the stamping and labeling facing inward and place into hybridization
tubes (Fisher 13 247 300) and clearly label the tubes. Multiple filters can be placed into the
same tube, up to about 7 per tube.
The pre-hybridization church buffer in the container can be reused
several times, so pour back into a 1L bottle.
á
Incubate in oven for a
minimum of 3 hours while labeling primers. Make sure the filters unroll so they lay against the sides
of the tubes. If not,
reverse the direction of the hybridization bottle.
á
Set up labeling reaction
(see Overgo worksheet)
á
To each hybridization
bottle, add denatured anchor probe and test probe. Typically, about 40ml of probe and 10µl of anchor. Let incubate overnight at 58¡C.
á
Before the washes the
next morning, for every two hybridization bottles, microwave one liter of 1.5X
SSC/ 0.1% SDS for 3 minutes and place into a 62¡C water bath until ready for use.
á
Empty the hybridization
solution into radioactive liquid waste and add 150ml 2X SSC/ 0.1% SDS to each
of the hybridization bottles.
Tighten the caps and place in the hybridization oven at 58¡C for 15 minutes.
á
While washing, place the
2 Pyrex Portables hot packs in the microwave for 3 minutes and 30 seconds. Then place in the bottom of the Pyrex
portable to heat the inside (see below if there is no shaking incubator
available).
á
Empty the 2X wash into
the radioactive liquid waste
á
With a long set of forceps
(Fisher 10 316B), pull the filters out of the hybridization bottles and into
the constructed Rubbermaid box. 13
is about the maximum number of filters per box.
á
For this next step we recommend a
shaking water bath at 58¡C. If this is not
available, an insulated container can be constructed that maintains the
temperature for the 30 minutes. Using a Clear
Magazine Snap Case (14Óx 8 5/8Óx 3 7/8Ó The Container Store 584070), cut
the two hinges from the top of the box so it will detach completely. Cut a piece of packing Styrofoam to
tightly fit into the lid. Place
the Styrofoam In a large Ziploc bag and wedge into the lid of the box. Take a 15ml conical
tube (Corning 430053) and cut in half. Cut a hole in the lid of the box that goes through the
Styrofoam and the Ziploc bag exactly the size of the conical tube (be sure it
is not any larger so the lip on the tube will prevent the tube from going all
the way through the hole. Next
slide the half of the tube with the cap through the hole all the way up to the
lid and seal around the tube with a glue gun. Get a funnel that fits in the conical tube. Lastly, get a 5 piece Pyrex
Portable with the hot packs
and place the box inside for insulation.
á
Pour a little 2X SSC/
0.1% SDS over the filters so they are a little manageable. Flip the filters one by one so they
face down trying to lay them stacked on one another. This will prevent sticking of the filters to one another so
they all get properly washed.
á
Place the lid on the box
and place inside the Pyrex Portable on top of the hot packs. Remove the cap on the conical tube and
place the funnel into the tube.
á
Pour the 1.5X SSC/ 0.1%
SDS into the funnel as quickly as possible to prevent the wash from cooling
down. Quickly remove the funnel
and replace the cap in order to zip up the container.
á
Place on an orbital shaker
(Fisher 14 512 24) for 30 minutes
á
As soon as you have the
2nd wash shaking, place the same amount of 1.0x SSC/ 0.1% SDS into
the microwave (about 3 minutes for 1L and 5 minutes for 2 liters) to heat just
above 58¡C
then place in a water bath set at 62¡C until ready to use.
á
Repeat wash with 1.0x
SSC using the same procedure as above
á
Cut a piece of blotting
paper to fit the inside of the imaging
cassette (Amersham RPN11643).
Tape the covered filter face up on to the filter paper. Place the imaging
screen (Amersham 63-0034-90) face down on to the covered filter and close
the cassette for exposure.
Filters from ChildrenÕs Hospital Oakland Reasearch Institute, should be
oriented with the pencil labeling across the top. Filters from Arizona Genomics Institute and Clemson
University Genomics Institute should be oriented with the serial number and
filter label in the bottom right corner.
There is a difference in stamping and this will compensate when scoring
the filters using Comboscreen.