Wash:             Sart time:______________
***Wash times can be shortened if necessary! I have done as little as 5’, but may depend on your probe. If you dry your membranes overnight, wash times may need to be increased.
 Pour out hyb buffer in radioactive liquid waste. Turn off temperature in hyb oven (set temp to 22℃).
 Add 50 ml 37℃ wash buffer, put hyb bottle in hyb oven for 1 min rotating but temperature off.
 Pour out into liquid waste, add 50 ml 37℃ wash buffer, rotate for 30 min in hyb oven (temp set @22℃). Temperature after 30 min:____________
 Repeat washes with 2 x 50 ml RT wash buffer, 30 minutes each.
-Tip. If background is still very high (as determined by Geiger counter) may want to continue washing blot at 37C.
 Take out blot with clean forceps into a tray with wash buffer. Drain off excess liquid and put blot in saran wrap. Squeeze out air bubbles and excess liquid. Tape edges. Put blot in film cassette, add positioning markers.
 Place blot in intensifier screen (these are great if you own one!), add Kodak MS film (greater sensitivity with intensifier screen) and expose blot at -80C.
 I usually do a 1-3 hour exposure to get an idea of how long I will need to expose. For really highly expressed miRNAs (i.e. miR-124, may only need 1 hour). For lowly expressed, may need several days. Alternatively, we also use a phosphorimager to image blots in lieu of film, which is preferred if you are planning to perform quantitation.

REAGENTS and SOLUTIONS
SequaGel (National Diagnostics cat# EC-833)
Temed (BioRad cat# 161-0800)
SafetyCoat: Baker cat# 4017-01
10% APS, Make a 10% w/v in DEPC treated H2O. Ammonium persulfate, Sigma A7460. Keep in refrigerator, make fresh every week.
10xTBE: (890 mM) 216g Trisbase Fisher BP 152-5 (MW 121.14)
(890 mM) 110g Boric acid Fisher (electroph.gr) BP 168-500 (MW 61.83)
(20 mM) 40 mL 0.5M EDTA pH 8.0
H₂O to 2L, check pH w. strip (take out and drizzle ~500 ul on
pH strip, ~pH 8.5, filter sterilize 0.2um, Fill 3 one liter bottles, mark level,
autoclave and bring up to level with H2O after cooling to RT

Fluka Bromophenol Blue-Xylene Cyonol solid mixture cat no 18047, when reconstituted to 5 ml, ~ 0.5% each dye in Tris-borate-EDTA buffer pH 8.3. Add 1.39 ml DEPC H₂O~1.8% dye. Mix 95 uL Formamide with 5 uL 1.8% Bromophenolblue/Xylene cyanolFF
Note: can use less dye.
Hybond N+: Amersham RPN303B
ULTRAhyb-Oligo Hybridization Buffer: Ambion cat # 8663, keep in refrigerator
T4 Polynucleotide Kinase: NEB M0201S
Gamma ³²P ATP, 6000 Ci/mmol, 150 mCi/mL PerkinElmer (cat# NEG035C005MC)
MicroSpin G-25 columns: Amersham 27-5325-01 (cellculture)
Wash buffer: 2xSSC/0.1%SDS
50 mL 20x SSC + 447.5 mL autoclaved H2O + 2.5 ml 20% SDS
This protocol has been put together with the help of many people from the McManus lab and surrounding labs at UCSF! Thanks for all of your help.

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