UV-Shadow and Semi-dry transfer:
 Take out all buffer from top reservoir with syringe. Un-clamp gel.
 Pry apart glass plates with spatula. Cut off upper right corner of gel. Place saran wrap on top of gel, flip over and remove other glass plate. (Be sure to wet the gel with a little TBE so it does not dry out and break!!)
 UV shadow gel with TC plate and take a picture to ensure samples are evenly loaded (although will eventually want to reprobe blot with a loading control).
 Put a piece of Whatman 3M paper (pre-wet with 1x TBE) on gel, put glass plate (or parafilm) on top and flip over.
 Measure gel and cut a piece of Hybond N+ to size (Lot#________________,) use clean gloves and scissors. Prewet in 1x TBE, position Hybond on gel, roll out bubbles with plastic sterile pipette. Put 1 piece of pre-wet (1x TBE) Whatman 3M paper on top. Transfer gel/membrane/whaman paper sandwhich to semi-dry transfer apparatus.
 Wipe off excess liquid around the edges of gel. Put lid on. Plug in the power supply and transfer at 250mA for 2 hours or overnight. Note: 300mA is the max on our power supply.

5’end-labeling of Probe:
 1 μl 10x PNK buffer (fresh from NEB)
 30 pmol probe DNA oligo
 1.66 μl [γ -³²P] ATP, 150 mCi/ml (= 250 μCi, 42 pmol) Vial#______________
 1 μl T4 Polynucleotide Kinase, 10 U/μl Lot#______________
 X μl Nuclease free H₂O to 10 μl
 37℃ at least 1 hr
 After incubation, bring reaction volume up to 50 μl before cleaning up with spin column! If too little volume is used, it will become trapped in column.

Microspin G-25 column:                  Lot#______________
 Resuspend the resin in the column by vortexing gently
 Loosen the cap 1/4 turn and snap off bottom, place in 1.5 ml screwcap eppendorf tube
 Pre spin column for 1 minute @ 735xg (start timer and fuge simultaneously).(Marathon 16K: 2900 rpm = 700xg, 3000rpm = 800xg)
 Place column in new screwcap tube and slowly apply sample to center of angled surface of resin bed. Do not disturb the resin.
 Spin column for 2 minutes @ 735xg. Discard column in radioactive waste.
 Take out 1 μl of labeled probe in 0.5 ml eppendorf tube, put tube in scintillation vial and count the cpm. Count:_______________

UV Crosslink:
 UV Crosslink the blot at 1200 x 100 μJoles after transfer.
 Optional: Allow membrane to dry overnight. Some evidence suggests this may improve sensitivity.
 Wet blot in 1xTBE, roll blot with transfer side in (upper right corner off) and put in hyb bottle. Try to get rid of all air bubbles between glass bottle and blot. Pour out any excess liquid.
Pre-Hyb: Ambion ULTRAhyb-oligo                Lot#______________
 Heat hyb buffer @ 65℃ briefly to dissolve any precipitated material, swirl bottle often.
 Take out ~25 ml (1ml/10cm²) in 50 ml conical tube, preheat @ 37℃.
 Add hyb solution to hyb bottle and pre-hyb for at least 5 min. Pre-hyb:___________

Hybridization:
 Pour out ~10 ml of pre-hyb into conical tube(keep “old” tube used to pre warm the hyb soln at 37 degrees) , add probe and mix, return to hyb bottle
 Hybridize O/N @37℃ (this incubation may be shortened to as little as 2 hours, but this should be optimized for each probe).
 Start hyb @ 37℃:________________ Stop:___________ Hyb time:__________

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