Bio-Rad PROTEAN II XL Cell - Manual (Page 24)

18
Section 6
Loading the Samples
Sample loading can be done in one of three ways. The most common method is to load liquid samples into
wells formed by casting a gel with a well-forming comb. The second method uses the entire gel surface as a
single well for liquid samples. The third method involves placing a tube gel or gel strip over the entire gel
surface, as in 2-D procedures.
6.1 Loading of Sample Wells
The approximate sample volumes that each well will hold for all available combs is included in
Section 13.2.
1. Prepare 1.5 liters of electrode buffer. Set aside 350 ml for the upper buffer chamber.
2. Pour 325-350 ml of electrode buffer into the upper buffer chamber. At this point, check the integrity of the
upper buffer seal. If the buffer appears to be leaking, remove the gel sandwich assemblies, re-lubricate the
gasket, and then re-attach the gel sandwich assemblies (as in Section 5).
3. Place the remainder of the electrode buffer into the lower buffer chamber. Lower the central cooling core
into the lower buffer chamber at a slight angle to prevent air entrapment under the gel sandwich. A few,
isolated bubbles under the gel will not affect the run. With 16 cm plates, it is necessary to dilute the lower
buffer with distilled water to a level of 1 cm above the bottom of the gel plates. Be sure to mix the lower
buffer well with a stir bar on a magnetic stirrer.
Note: Dilution of the lower buffer by up to 1:2 with dH
2
O will have no detrimental effect on resolution. Dilution
of the upper buffer is not recommended.
4. Load the samples into the wells under the electrode buffer with a Hamilton syringe. Insert the syringe to
about 1-2 mm from the well bottom before delivery. Disposable pieces of plastic tubing may be attached
to the syringe to eliminate the need for rinsing the syringe between samples.
Note: The sample buffer must contain either 10% sucrose or glycerol in order to underlay the sample in
the well without mixing.
5. An easier method of sample loading is to use an Eppendorf-like pipettor and disposable tips. To accomplish
this successfully, use the optional beveled short plate (catalog number 165-1827 for 16 cm and 165-1828
for 20 cm) so that you can insert the pipet tip further into the well before sample delivery. This will prevent
inter-well mixing of samples. Or, use the Bio-Rad Prot/Elec
™ tips (catalog number 223-9915 or 223-9917),
which are designed for loading samples into wells.
6.2 Loading a Single Sample Per Gel
In this procedure, a gel is cast without using a comb, forming a flat gel surface. This gel is cast with an
overlay solution. This type of sample application can be used for preparative pur poses, but it is most often
used in blotting applications. After electrophoresis, the sample is transferred electrophoretically to a
membrane, which then can be cut into several identical strips for analysis. Follow the instructions for casting
the separating portion of a discontinuous gel (Section 4.1), except pour the gel nearly to the top of the
sandwich. Allow just enough room for sample loading. (A stacking gel may also be added to this type of gel.)
1. Prepare electrode buffer, and add to lower reservoir as in Section 6.1. Place the central cooling core in the
lower chamber, and add electrode buffer to the upper reservoir chamber.
2. The sample may be loaded with an Eppendorf-type pipettor, with a needle and syringe, or with a Hamilton
syringe. Start at one end of the gel, and deliver the sample gently and evenly over the entire length of the
gel. Layer the sample as closely as possible (1-2 mm) to the surface of the gel.
6.3 Gels as Samples
All two-dimensional techniques involve this variation of sample loading. Either a cylindrical gel or a strip of a
slab gel may be placed on top of a slab gel for separation into a second dimension. This procedure is described
in detail in Section 10.