Northern Blot
Ⅰ.Label probe
1. preparation labeling of probe -> total 34㎕ (prepare 3 E-tube)
-22~23㎕ dH2O
-10㎕ random primer
-25ng (1~2㎕) DNA
2. In RI room
set - incubator @ 68℃, warm hybridization buffer
- heat block @ 37℃
- turn cooker on (max temp)(boiling)
- place RI @ RT
3.boiling mixed probe (34㎕) 5min -> spin down lightly
4.lay WYPALL,
add - 10㎕ 5x dCTP buffer
- 5㎕ 32P
- 1㎕ Exo(-) Klenow (5v/㎕) ->pipetting
5. incubate labeled probe at 37℃ heat block 10min
Ⅱ. Pre-hybridization
1. input 5㎖ of hybridization buffer in a sealing bag
** pouring into backside of membrane. Don't touch membrane to the utmost
2. remove bubble -> 68℃ agitation (>15min)
3. put membrane upside down to completely remove bubble
Ⅲ. Filtration with column
** during that 10min, fill STE buffer in purification column using syringe.
** This procedure must be sequenced.
1. remove cap of column
2. put 1x STE (stored 4℃) 80㎕
3. as pulling a piston, drive a screw of syringer at column
4. push a piston with feeling of solution move down. If leak.. it's OK!
5. put column RT as it stand until probe incubation finish
6. after 10min, add 2㎕ stop mix to probe
7. add probe (50㎕) to column
8. push a piston and collect flow-through to used E-tube
9. add 40㎕ STE buffer to column, collect to new E-tube
10. counter RI (100x)
Ⅳ. Hybridization
1. boil the probe 5min -> chill on ice 2min
2. add 20㎕ probe to 600㎕ hybridization buffer into a new E-tube
3. membrane: pour prewarmed buffer a little, add '2'mix to that,
4. fill hyb buffer,sealing and hybridize 68℃ with agitation for 2hr
Ⅴ. Washing
1. warm wash solutionⅡ at 68℃, turn 37℃ incubation on
2. pour wash solutionⅠ in plasic case
3. at waste room, carefully pour out, take membrane out with a pincette
4. soak wash solutionⅠ, incubation 37℃ 10min
5. during that time, prepare WYPALL and aluminium foil
6. after 10min, put membrane on foil
7. detect RI (100x)
8. discard wash solutionⅠ, repeat (pour Ⅰ again, 37℃ 10min-> detect)
9. soak membrane in warmed at 68℃ wash solutionⅡ
10. 37℃ incubation 10min, detect, repeat (W.SⅡ)-> detect
11. cover membrane with OHP film, put cassette, fix with tape
12. in dark room, cover a film on membrane -fit edge
Ⅵ. Exposing & Developing
1. expose -70℃ overnight
2. develop with develop machine |
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