C.B.S. Scientific DH Series - Manual (Page 20)

C.B.S. œ Scientific
20
Dedicated Height Sequencer
B. Transfer of gel onto blotting paper-continued
2. Place a sheet of pre-cut (slightly larger than gel) blot/chromatography paper to fit on
top of the gel and press down firmly and evenly over the entire gel surface. The gel
will come off the glass and stick to the paper as you lift the paper/gel sandwich slowly
and carefully away from the glass plate. NOTE: At this step, if you were using 35S
you could follow the alternative fixing procedure given if greater band resolution is
desired.
3. Place paper/gel sandwich on top of a perforated plate or screen and place the entire
assembly over a large tray or basin to catch fixative.
4. Slowly pour over the entire surface of the gel, a solution of 10% acetic acid and 10%
methanol. Allow to soak for 15-30 minutes, then tilt to drain.
5. Cover gel evenly with plastic wrap. Invert or flip to remove the screen and to secure
the excess plastic wrap on the backside of the gel (paper side). Avoid trapping
bubbles between plastic and gel. Bubbles can cause distortion on the autorad. If
necessary, cut the gel to fit the gel dryer. NOTE: Usually the top 10cm of the gel can
be removed since there is no readable DNA sequence in this region of the gel.
6. Place paper/gel on gel dryer and dry for 30-65 minutes with 80
°C heat and under
vacuum.
7. Place dried gel in X-ray cassette and expose to X-ray film for 12 to 16 hours for 32P
(@-20
° to -70°C; using 2 Bio-Speed intensifying screens) and 24 to 48 hours for 35S
@ room temperature using only 1 intensifying screen.
8. Develop/process autoradiogram manually or in an automated film processor and read
DNA sequence (CBS Personal Gel Reader, cat. # DQX-4000) on a light box (CBS cat.
# SQI-1104).
C. Fixing the gel directly on the glass plate
1. Pry apart glass plates as in step B-1. Place glass plate/gel assembly on top of a large
tray and proceed with steps 6-9.