Buffer chamber

A schematic diagram of the apparatus is shown in Figure 3.2. The molecules are introduced under a partial vacuum of 10 torr into a buffer chamber that communicates via molecular slipstream with the source itself at 10 to 10 torr in order to ensure a constant concentration in the source at all times during the analysis. 

Figure 3. Illustration of the cross-beam machine. N is the nozzle source for the molecular beam, C is the buffer chamber with a beam chopper (not shown), H is the hexapole electric field quantum state selector, U are the homogeneous electric field plates, Q is an on-axis quadrupole mass filter, O is the fast atom beam source, and Q and C,8o are channeltrons.

Put the plate on the cooling plate of a horizontal electrophoresis apparatus. Fill the buffer chambers with 1 1 diluted Soln. B (= electrode buffer) and connect the electrode buffer to the gel by filter paper bridges, wetted with electrode buffer. The filter paper covers the gel for about 5 mm. Remove carefully air bubbles between papers and gel. 

FIGURE 3.2 A slab gel electrophoresis apparatus. A voltage source generates an electric potential difference between the upper and lower buffer chambers, causing the applied DNA sample to migrate through the gel toward the positive electrode. 

Run the slab gel in a vertical dimension. The current should be set at 25-30 mA per slab gel. Allow the electrophoresis to proceed until the tracking dye is at the bottom of the gel. Turn the power off and remove the gels from the buffer chambers. Carefully separate the sandwiched plates with a thin spatula and transfer the gel in one piece to a tray containing staining solution. 

Depending on the design of the gel apparatus, it may be preferable to load the gel following installation of the gel into the apparatus and addition of buffer to the upper buffer chamber (steps 23 to 25). 

Fill lower buffer chamber of the electrophoresis apparatus with SDS-PAGE tank buffer. 

Carefully fill upper buffer chamber with SDS-PAGE tank buffer. Do not pour buffer into the sample wells because it will wash the sample out. 

The cathode (black lead) is connected to the upper buffer chamber. 

Run the gel (see Basic Protocol 1, steps 23 to 30), but fill the lower and upper buffer chambers of the electrophoresis apparatus with lower (anodic) and upper (cathodic) Tris-tricine tank buffers, respectively, and adjust current to 70 mA per gel. 

Add thioglycolate to the electrophoresis buffer in the cathode buffer chamber to a final concentration of 0.1 mM (11.4 mg per liter buffer). 

The accuracy of BE-AES measurements is directly related to the accuracy with which a solution concentration can be determined. ICP-AES, requires milliliter quantities of sample to accurately determine a concentration. However, the technique is sensitive to low micromolar concentrations of most elements. The high analyte concentrations obtained during the final concentrating step are necessary for maximizing the difference in concentrations between the two buffer chambers and as such must be diluted prior to ICP-AES concentration determination. Table 18.1 indicates approximate concentrations and volumes present during buffer equilibration and ICP-AES concentration determination. Unless otherwise noted, concentrations are the actual concentration in the buffer equilibrium step prior to dilution for ICP-AES measurement. 

Add 0.5X TBE buffer to the upper and lower buffer chambers. Remove the comb from the gel. Connect the negative electrode (cathode) to the top buffer chamber and the positive... 

Reconnect the power supply and apply 200 V (constant voltage). Continue the electrophoresis until the bromophenol blue (dark blue) dye just migrates off of the bottom of the gel into the lower buffer chamber. Any unincorporated radiolabeled nucleotide that was present in the samples will run off of the bottom of the gel with the dye. Remember that the buffer in the lower chamber is now radioactive. 

High voltage electrophoresis (2000-4000 V) on Whatman 3 MM paper (57 cm in length) is carried out in an apparatus in which the ends of the paper are immersed in the two buffer chambers and the rest of the paper is supported on a rack that is completely immersed in an inert solvent (e.g. Varsol), cooled by circulating tap water. In our laboratory we use a Savant apparatus, but others are also suitable. 

Pour one 1% agarose gel in TAE buffer for every 24 DNA samples. Use 110 mL of agarose mixture for each leveled gel apparatus (12 x 15 cm surface area). Insert two 14-place, 2-mm combs one at the top and one in the middle of the gel. Allow gel to harden (15-20 min) and carefully remove the combs (see Note 5). Flood the gel and buffer chamber with enough TAE buffer to cover the gel with approx 0.5-1.0 cm of buffer. 

After polymerization of the gel, remove the comb and rinse the sample wells with water. Place the gel into the electrophoresis apparatus. Fill upper and lower buffer chamber with IX TBE so that the wells are submerged. 

The combination of two plasma polymerization chambers divided by an evacuated buffer chamber allows two different gases to be introduced into each chamber with and/or without initiation of a plasma. The pressures in each chamber are independent of that in the other and there is no cross-contamination of gas thanks to the buffer chamber. Possible combinations of substrate treatments are ... 

Place gel in gel apparatus and add cathode buffer and anode buffer to upper and lower buffer chamber, respectively. 

Put about 400 mL of buffer into each side of the buffer chamber or until it reaches the leveling mark. 

Notice that you cannot put your hands into this conducting solution unless the power is off. This is a safety feature and a good one. Carefully remove the upper chamber and take a close look at it. This is called the upper buffer chamber. Notice that it has 18 rubber-stoppered ports in it, so 18 samples can be done at the same time. If you are only going to do two samples, then the rest of the ports are plugged with rubber stoppers. If any glass tubes have been left in the apparatus, carefully remove them. 

Your sample tubes are placed where the rubber stoppers are and go down into the lower buffer chamber. Practice removing the plugs. Only the red rubber portion is removed. Pull it out from the bottom and then put it back by twisting it a little and then finally pushing it in with a glass stirring rod. 

You want to maintain electric equivalence, so you do not use holes next to each other. Use holes opposite to each other as much as possible. Examine the lower buffer chamber. It has a water jacket around it so that the samples can be cooled if necessary. The upper buffer and the lower buffer are separated as stock solutions, because the upper buffer has a marker dye in it that the lower one does not have. These buffers can be used again. Notice on the buffer stock solutions that the label tells how many people have used them. You can use this solution four different times before you have to change it. Please initial the label, showing that you have used it. Fill the lower buffer chamber to within 1 cm of the rim. 

Examine the lower buffer chamber. It has a water jacket around it, so that the samples can be cooled if necessary. Fill the lower buffer chamber to within 1 cm of the rim with 0.05 M acetic acid. This aids in cooling. 

Electrophoresis can be carried out in different types of electrophoresis cells. For geometric reasons, these cells have to be miniaturized and they must be cylindrical. They consist of two concentric chambers. An outer glass or plastic centrifuge tube forms the anodal buffer chamber (cf. Fig. 8.2(a)). The volume of the buffer chambers must be large enough to minimize the increasing cell resistance. 

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