C.B.S. Scientific EBU-204 - Manual (Page 8)

8
EBU-204 Mini-Blotting System Instructions 6-2-14
SECTION 3
Instructions for Use
3.1 Blotting Unit Preparation
1 Place the blotting chamber on a level work surface in an authorized work area.
2 Consult applications table 4 1 for type of transfer, buffer system, membrane type
and power settings
Coolant Circulation:
For coolant circulation, connect a heavy wall tubing which will not kink (hose clamps
are optional), to the tubing adapters supplied with the unit Excessive pressure will
damage the plexiglass in the base and create leaks Attach a regulated pump or
recirculating water bath (follow manufacturer’s instructions), recommended flow is
500 ml per minute or 15psi Do NOT use tap or house water as it normally delivers
too much pressure and can be subject to large fluctuations in pressure.
1 Place, do NOT drop, a magnetic spin bar (not supplied by C B S ) in the bottom
of the tank This maintains pH, buffer and heat circulation during the electroblot
2 Place tank on top of the magnetic stirrer
3 Connect to a cold water supply (see coolant circulation above)
4 Buffer should be pre-cooled to 10°C
3.2 Preparing the Gel for Transfer
The EBU-204 can hold up to 4 gel cassettes simultaneously with space provided for
a magnetic stir bar to allow buffer circulation and heat exchange for uniform transfers
When the resolving gel electrophoresis is complete, proceed with staining and photo-
documentation if applicable Cut one corner off the gel so that correct orientation is
maintained throughout the procedure
Preparation of Electroblotting Components
1 CBS membranes are supplied with the blotting paper cut to the correct size for
CleaPAGE gel transfers. If not using CBS membranes, cut transfer membrane to size
of gel or sufficient dimensions to cover the relevant bands. Also, pre-cut Whatman-
type 3MM filter paper to same size and soak in transfer buffer until completely
saturated (15 to 30 minutes)
2 Pre-wet membrane chosen according to manufacturer’s instructions Nylon or Nylon-
supported nitrocellulose should be soaked in ddH
2
O PVDF in MeOH Equilibrate all
types in transfer buffer for 3 minutes Submerge a single foam pad and gently tease to
release trapped air bubbles until pad is completely saturated
3 Pre-equilibrate gel in 1x transfer buffer to be used prior to electroblotting for 5 to 30
minutes depending on gel thickness
4 Open the transfer cassette as shown below in figures 1-2 and lay it flat on the bench.