C.B.S.
Scientific Co, Inc.
10
Semi-Dry
Blotters
d. Apply a saturated membrane on the top of the paper.
e. Carefully place the gel on the membrane, making sure no bubbles remain
between the gel/membrane interface.
f. Stack the remaining 3-4 sheets of blotting paper on the top of the gel. Add
one sheet at a time and be sure to ‘smooth’ with glass rod to remove air
bubbles.
g. Close the lid which contains the cathode (-). Holding the two white nylon
thumbscrews, place the lid over the stack and secure while applying some
downward pressure (gently!).
4.
Power Settings
a. Calculate power input by using 0.8mA/sq. cm of gel for larger gel transfers.
Mini-gels (10 x 11cm) may be transferred at up to 100mA (5-25V) total
current. Remember, smaller proteins (<20kd) require less time than medium
size (<80kd) or larger proteins.
b. At the conclusion of the run, turn power supply off and disconnect leads from
blotter. Cover will not release unless power cord is removed. Remove the
cover slowly to catch any part of the gel sandwich sticking to the cathode.
After each use, clean the electrodes of buffer salts by rinsing in distilled
water.
C.
Semi-Dry Blotting of RNA (Northern)
1. Electro-Blotting RNA
a. Buffer System:
1X TAE (40mM Tris-acetate, 1mM EDTA, pH8.0)
4.84g
Tris-base
1.14ml glacial acetic acid
0.37g
Na2EDTA-2H
2
O
pH to 8.0 with HOAC
or
1XTBE: (89mM Boric Acid, 2.5mM EDTA, pH 8.4)
10.8g
Tris-base
5.5g
Boric
Acid
0.93g
Na2EDTA-H
2
O
H
2
O to 1 liter
2. Preparation of the Gel
a. Formaldehyde must be removed from the gel by soaking in 0.1XTAE for at
least an hour at room temperature.
b. Standard electrophoresis buffers for RNA are usually 1XTAE or 1XTBE.
Electro-blotting transfer occurs in low ionic strength buffer. Exchange the 1X
electrophoresis bufferby soaking in 0.1X buffer for 20-30 minutes.
3. Preparation of the Apparatus