blotting
1. Run gel to desired distance.
2. Visualize gel on UV box. Note the marker bands by:
a) using a fluorsecent ruler and photographing the gel; or
b) marking the bands directly on the gel by punching a small hole at each band with an 18 guage hypodermic needle then lightly marking each hole with a dab of loading dye when gel is on the blotter
3. Soak gel in 0.2 N HCl until the bromophenol blue band begins to change color. (Around 10 min usually). Alternatively, expose to UV
transilluminator for 90 sec.
4. Rinse once with H2O.
5. Soak in 0.4N NaOH/0.6M NaCl for 30 min.
6. Immediately place gel on blotting apparatus:
7. Blot at least 8 hours (better overnight) with 10X SSC.
8. Remove membrane from blotter. Discard gel, whatman, and paper towels.
9. Rinse membrane in 2X SSC. Blot dry on a piece of whatman.
10. Sandwich blot between two pieces of dry whatman and bake in vacuum oven for 1 hour.
prehybridization
1. Place blot into a hybaid tube with the DNA side facing inward.Fill tube with 2X SSC and then gently pour out to ensure that no air bubbles are trapped between the blot and the glass.
2. Add 10 ml of Prehybridization Solution and incubate on a roller at 42℃ for at least 1.5 hours. Overnight is okay but don't go longer than about a day and a half because background will increase.
hybridization
probe synthesis
Prepare probe fresh every time and do this while blot is prehybridizing.
1. Use 20 to 100 ng of probe DNA, preferably in as small a volume as possible.
2. Add H2O to probe to bring volume to 9λ. Boil 5 min. then ice for 1 min.
3. Add 2λ 10X reaction buffer/hexanucleotide mix
3λ nucleotide mix without dCTP (-dCTP mix)
5λ 32P α-labelled dCTP
1λ Klenow
blotting
1. Run gel to desired distance.
2. Visualize gel on UV box. Note the marker bands by:
a) using a fluorsecent ruler and photographing the gel; or
b) marking the bands directly on the gel by punching a small hole at each band with an 18 guage hypodermic needle then lightly marking each hole with a dab of loading dye when gel is on the blotter
3. Soak gel in 0.2 N HCl until the bromophenol blue band begins to change color. (Around 10 min usually). Alternatively, expose to UV
transilluminator for 90 sec.
4. Rinse once with H2O.
5. Soak in 0.4N NaOH/0.6M NaCl for 30 min.
6. Immediately place gel on blotting apparatus:
7. Blot at least 8 hours (better overnight) with 10X SSC.
8. Remove membrane from blotter. Discard gel, whatman, and paper towels.
9. Rinse membrane in 2X SSC. Blot dry on a piece of whatman.
10. Sandwich blot between two pieces of dry whatman and bake in vacuum oven for 1 hour.
prehybridization
1. Place blot into a hybaid tube with the DNA side facing inward.Fill tube with 2X SSC and then gently pour out to ensure that no air bubbles are trapped between the blot and the glass.
2. Add 10 ml of Prehybridization Solution and incubate on a roller at 42℃ for at least 1.5 hours. Overnight is okay but don't go longer than about a day and a half because background will increase.
hybridization
probe synthesis
Prepare probe fresh every time and do this while blot is prehybridizing.
1. Use 20 to 100 ng of probe DNA, preferably in as small a volume as possible.
2. Add H2O to probe to bring volume to 9λ. Boil 5 min. then ice for 1 min.
3. Add 2λ 10X reaction buffer/hexanucleotide mix
3λ nucleotide mix without dCTP (-dCTP mix)
5λ 32P α-labelled dCTP
1λ Klenow
4. Mix gently and incubate at 37℃ for 1-1.5 hr.
5. Add 50λ 1X TE pH 8.0 to probe and transfer whole volume to a Sephadex G-50 column or a MicroSpin S-200 column
6. Centrifuge:
S-200 column: 2 min at setting 3 on microfuge
G-50 column: 5 min at 1000rpm in Sorvall or tabletop
7. Collect flowthrough. Check activity of labeled probe then discard column.
8. Flash spin and add to hybridization solution. Boil 10 min then add to membrane
hybridization
1. Add probe to 10 ml prehybridization solution.
2. Discard prehyb and replace with hybridization solution containing probe.
3. Incubate on roller at 42℃ for at least 8 hrs. I usually hybridize overnight.
washing
1. Fill hybaid tube containing blot to about 3/4 full with 2X SSC. Securely cap and invert a few times. Discard wash into 32P liquid waste container.
2. Remove blot from tube and place into Tupperware container with 250ml 2X SSC/0.1% SDS.
3. Perform following series of washes and check blot at every step:
2X SSC/0.1% SDS RT 15 min.
1X SSC/0.1% SDS RT 15 min.
1X SSC/0.1%SDS 65oC 15 min
0.5X SSC/0.1% SDS 65oC 15 min.
0.1X SSC/0.1% SDS 65oC 15 min
4. Remove blot from wash. Rinse in 2X SSC at RT briefly and then wrap damp blot in saran wrap.
5. Place on film at -80℃ with an intensifying screen or place on PhosphorImager screen.
SOLUTIONS
0.2N HCl
8.3 ml concentrated HCl (12.1N)
H2O to 500 ml
0.4N NaOH/0.6M NaCl
20 ml 10N NaOH
60 ml 5M NaCl
H2O to 500 ml
prehybridization/hybridization solution
For 100ml add (in this order):
50 ml formamide
25 ml 0.5M NaH2PO4 pH7.2
15 ml H2O
5 ml 5M NaCl
7.0 g SDS
Dissolve and store at 4℃
Wash solutions
