NORTHERN BLOT PROTOCOL1

NORTHERN BLOT OF RNA GEL

Buffers:

20XSSPE (500 ml):
3.6M NaCl : 105.2 g
0.2M phosphate buffer pH 7.0: 100mL of 1M
20mM EDTA: 20mL of 0.5M

Phosphate buffer pH7.0 (500ml):
NaH2PO4 (monobasic) 140 mL of 1M
Na2H2PO4 (Dibasic) 360 mL of 1M
pH to 7.0 after both are added

Hybridization buffer =HB (100mls):
5X SSPE: 25mls 20X
2% SDS: 20 mls 10%
**Right before using add
1000ug denatured RNAse free CT DNA (heat to 95o for 3 min to denature)
5000ug yeast RNA

WASH:
2X SSPE + 0.1% SDS (500 mls)
50 mls 20X
5 mls 10% SDS= 0.1% SDS

Running buffer (1L):
100 mls 10X MOPS
52.6 mls of formaldehyde
847.4 mls H20

RNA Gel (70ml):
0.84 g agarose
7 mls 10x MOPS
58.38 mls H2O
put gel in solution by heating
let cool to 60°C
add Formaldehyde to equal 0.66M --3.78 mls
pour gel, in hood if possible

Run gel
after, wash gel with distilled water
put on posiblotter

layers from top to bottom:

sponge
3-4 Whatman paper cut to size
( both of these up above should be soaked in 10X SSPE but not dripping)
gel
mask --make sure the gel overlays the mask so that it can seal
membrane with line of the top of gel and the right top corner marked
3 pieces slightly bigger 3M paper
hard plastic with holes
clamp on and make sure there is a good seal, pressure 75-80 mm Hg for1hr or more
blot membrane dry, cut top right corner
wrap in saran wrap and UV irradiate, RNA side down for 2.5 min
wash couple min in hot 2X SSPE/0.1% SDS sol'n (microwave 30-45sec, 50% power)
air dry

Pre Hybridize, 6 hrs-overnight:
set oven to 65°C
heat HB buffer to put SDS in solution
roll up membrane inside piece of mesh and put into hybridizaton tube(2XSSPE/0.1%SDS)
check for air bubbles
put tube in oven balanced with another tube on opposite side

Labeling probe High Prime (BMC):
1) put 25ng of probe in a total volume of 11uL with ddH2O
2) denature @ 95°C 3 min.
3) quick cool on wet ice
4) add 4ul High Prime (Boehringer Mannheim)
    5ul alpha P32 dCTP 3000uCi/mmole
    20ul total
5) incubate 10-15 min 37°C
6) add 28uL of 1X TE, 2ul 0.5M EDTA, to 20ul reaction
7) prespin G25 column for 1 minute
8) Add 50ul sample to column and spin for 2.5 min in clinical centrifuge
9) throw away column
10) mix in a new tube 100ug CT-DNA
    50ug Yeast RNA
     in a 0.5mL tube
12) denature 95° 3 min
13) add to hybrid tube mix, hybridize at 65°C for 20-36 hrs

Post hybridization:
hybridize for 36-48 hrs
wash
1) heat up 2X SSPE/0.1% SDS (wash) heat up to 65° C (?use microwave?to warm)
2) take out tube
3) pour liquid iinto hot radioactive waste
4) rinse with 10ml wash do this twice and pour waste out
5) put in 40 to 50 mls of wash and shake vigoursly
6) put in oven for 20 min rotating
7) pour down sink
8) another 40-50mls and shake, oven
9) pour down sink and make sure it is no longer hot and then take out membrane
10) wash on shaker with 0.2XSSPE w/ 0.1% SDS at RT for 15 min.
11) air dry, expose yourself