Biology Reference
In-Depth Information
3. 50
Denhardt
'
s solution
Ficoll (Type 400)
0.1 g
Polyvinylpyrrolidone (PVP)
0.1 g
Bovine Serum Albumin (BSA)
0.1 g
DEPC-ddH
2
O
to 10 mL
Dissolve components, pass through a 0.45
m
m filter, and store in 200
m
L
aliquots at
20
C.
4. Salmon sperm DNA
Lyophilized DNA
1 g
Tris-EDTA pH 8.0 (TE, Fisher #BP2473-1)
100 mL
Add TE to DNA in an autoclavable glass Wheaton bottle. Autoclave (121
Cat
15 lb/in
2
for 15 min), cool to room temperature, dispense into Eppendorf tubes
in 1 mL aliquots, and freeze at
20
C. As a result of autoclaving, the DNAwill
have been sheared to the 200 bp-5 kbp range, which can be checked by running
an aliquot on an agarose gel.
5. Prehybridization buffer
20
SSC (Fisher, #BP1325-1)
2 mL
Dextran Sulfate
1 g
Formamide
5 mL
0.5 M EDTA
40
m
L
50
Denhardt
'
s Solution
200
m
L
Sheared Salmon Sperm DNA
1 mL
Add DEPC-ddH
2
O up to 10 mL. Vortex the suspension and heat to 65
C to get
into solution. This may take 30 min or more.
6. Formamide buffer (FB)
Formamide
30 mL
20
SSC
0.5 mL
Add up to 50 mL with DEPC-ddH
2
O in sterile plastic Falcon tube or equivalent.
7. Tris pH 7.5, NaCl (TN), and TN-EDTA
1M Tris, pH 7.5 (Fisher, #1757-500)
50 mL
2M NaCl
37.5 mL
Add up to 500 mL DEPC-ddH
2
O and autoclave.
For TN-EDTA, add 1 mL of 0.5M EDTA to 50 mL of TN.
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