C.B.S.
Scientific Co, Inc.
7
Semi-Dry
Blotters
SECTION 3
Instructions for Use ---
The instructions are divided into the following three categories:
A. General
Considerations
B. Semi-Dry Electro-Blotting of DS DNA from Agarose Gels (Southern)
C. Semi-Dry Blotting of Proteins (Western)
D. Semi-Dry Blotting of RNA (Northern)
3.1
Blotting Unit Preparation
Place the blotting chamber on a level work surface in an authorized work area.
A.
General Considerations
•
Because transfer efficiency depends on many factors (e.g. gel concentration and
thickness, size, shape and net charge of molecule) results may vary. For resolving
gels, the % acrylamide range from 8 to 10% will separate with close to 100%
efficiency the transfer of 14 – 66 kD.
•
Polyacrylamide gels have pore sizes that are too small for effective capillary diffusion
of DNA. These types of gels must be blotted electrophoretically requiring a transfer
buffer of low ionic strength (0.5 to 1X TBE). This is why nylon membranes are
generally used (uncharged nylon being preferable to positively charged nylon).
Nylon is the membrane of choice because it will bind DNA fragments as small as
50bp and can be irradiated to covalently linking the DNA to the membrane.
•
The general “rule of thumb” for electrophoretic transfer in semi-dry blotting units is
application of a maximum current of 0.8mA/cm
2
of gel area. For gels used in the
EBU-4000 (15x15cm up to a max. of 20x20cm) this translates to upwards of 300mA.
In the larger EBU-6000, this can increase to 1200mA. Please ensure that an
adequate power supply is being used to power the transfer.
•
In general, most runs should take between 15 to 60 minutes depending on the size
and type of the gel and they type of transfer, i.e., Southern, northern or western.
•
After setting the current at mA range, one can usually switch to the voltage panel to
obtain the voltage being applied. Most power supplies allow you to switch back and
forth between the amperage and voltage.
•
Percent of acrylamide
Size range transferred
(resolving gel)
(approx.
100%
efficiency)
5-7
29-150
kD
8-10 14-66
kD
13-15 <36
kD
18-20
<20
kD
