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Measure concentration 2uL of RNA with 298uL of TE in BioSpec.
Tape gel box and bath in hydrogen peroxide for 30mins.
Rinse with RNASE free water.
Make 1x MOPS for gel boxes
Make fresh 2x sample buffer
a) 150uL 10x MOPS
b) 263uL Formaldegyde
c) 750uL Formamide
d) 10uL 10mg/mL EtBr
6) Make gel. Dissolve 1.05g of RNase free agarose (aquaPor LE) in 60mL of RNase
free water in flask.
7) Heat.
8) Then add 7mL of 10x MOPS and 3.5 mL of formaldehyde.
9) Pour into gel tray immediately under fume hood.
10) Prepare RNA samples and marker. (Make sure to have enough. 20uL/well/gel).
11) Use 10mg of RNA, DEPC water, and 10uL of 2x sample buffer
12) Incubate samples @65o for 15mins. Chill at 4o.
13) Add 2uL of loading buffer
a) 50% glycerol
b) 1mM EDTA, pH 8.0
c) 0.25% bromophenol blue
14) Spin briefly
15) Load gel with marker and samples (20uL/well).
16) Run at 70-80V until blue dye is ¾ of the end of the gel in 1x MOPS.
17) Wash glass transport trays with RNase Free water.
18) Take picture in UV light box with saranwarp cover and RULER
19) Wash gel with 2x10mins in RNase free water at RT.
20) Prepare turboblotter apparatus.
a) Place 1.5 inches of thick blotting paper
b) 3 thin dry Whatman paper
c) 1 thin Whatman soaked in 10x SCC
d) Hybond-N+ nylon membrane soaked in RNase Free water for 10mins.
e) Place gel on top of member and mark orientation with grease pencil.
f) Pour a small amount of transfer buffer onto gel.
g) 3 thin Whatman soaked in transfer buffer.
h) Remove air bubbles between gel and member by rolling pipet.
i) Add top tray and fill moat with transfer buffer.
j) Place plastic cover on top.
k) Blot overnight.