Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 16)

8. Using a 50 ml conical centrifuge tube, add 5 ml of lyticase buffer (10 mM Tris, pH 7.2, 50
mM EDTA, 1 mg/ml lyticase) for each 1 ml of plugs. Push the solidified agarose plugs,
using the snap off tool provided on the plug mold, into the 50 ml centrifuge tube contain-
ing the lyticase buffer. Incubate the plugs 30 minutes to 1 hour at 37 °C without agitation.
9. Remove the lyticase buffer and rinse the plugs with 25 ml of 1x wash buffer (see step 10
for wash buffer recipe). Add 5 ml of Proteinase K Reaction Buffer (100 mM EDTA, pH
8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, 1 mg/ml Proteinase K) for
each ml of agarose plugs. Incubate the plugs overnight at 50 °C without agitation.
Note: various cell lines have been incubated up to 4 days in Proteinase K without detri-
mental effects to the quality of DNA.
10. Wash the plugs four times in 50 ml of wash buffer (20 mM Tris, pH 8.0, 50 mM EDTA),
30 minutes to 1 hour each at room temperature with gentle agitation. If the plugs are to
be used in subsequent enzymatic reactions, wash the plugs in 1 mM PMSF during the
second or third wash to inactivate any residual Proteinase K activity.
11. Store the plugs at 4 °C. The plugs are stable for 3 months to 1 year.
12. Maintain the plugs in 1x Wash Buffer for long term storage. However, for subsequent
restriction digestion, the EDTA concentration must be lowered. Wash the plugs to be
restricted for 30 minutes in 0.1x wash buffer or TE. See Section 3.6 for more information
on restriction digestion of plugs.
3.6 Restriction Enzyme Digestion of Plugs
1. Place one plug per digest in a sterile 1.5 ml microcentrifuge tube. Incubate the plug with
1 ml of the appropriate 1x restriction enzyme buffer for about 1 hour with gentle agita-
tion at room temperature. Aspirate off the buffer and add 0.3 ml of fresh 1x restriction
enzyme buffer. Add the restriction enzyme (30-50 U per 100 µl plug) and incubate
overnight at the appropriate temperature.
Note: Some restriction enzymes require shorter incubation times for complete digestion
(2-4 hours). This should be determined empirically.
2. After overnight digestion, remove the buffer and add 1 ml of wash buffer.
Note: If the plugs are to be stored for more than 1 day, remove the wash buffer from the tube
and store at 4 °C. This will prevent possible diffusion of small (<100 kb) DNA fragments
out of the agarose plug.
3. Load 1 ⁄
3
to 1 ⁄
2
of a plug (approximately 2 mm) per well and adjust the volume if necessary
on subsequent gels. In addition, always load appropriate size standards.
Note: For a 10 mm wide well use 1 ⁄
2
of the plug (10 mm x 2 mm). For a 5 mm wide well
use 1 ⁄
3
of the plug (5 mm x 2 mm).
3.7 Hemocytometer Usage
A hemocytometer is usually divided into nine large squares (Figure 3.1). Each large square
is 1 x 10 -4 cm 2 or 0.1 mm 3 ; one such square (A) is shown the figure with darkened borders.
The large circle around the center square (B) represents your field of view at 100x power
(10x objective lens, 10x eye piece). The center square (C) is subdivided into 25 smaller
squares. The smaller circle in the center square represents your field of view at 400x power
(40x objective lens, 10x eye piece). These 25 center squares are further divided into 16 squares.
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