N and L Series Buffer Preparation for Normal Processing of Nucleobond AX Cartridges
The most important factors when making up the N or L series of buffers for Nucleobond AX cartridges are the concentration of KCl and the pH. Even slight deviations in these could change the elution profiles of different nucleic acids. The following guidelines are presented primarily for those laboratories who use Nucleobond AX cartridges with buffers that they choose to make themselves. The protocol assumes that the buffers and cartridges will be used for both DNA and RNA purifications. If only DNA is to be purified, the DEPC treatment in step 1 can be omitted. The following procedure will make one liter of each buffer.
NOTE 1: The ethanol and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the ethanol or acid approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.
- Prepare DEPC (diethylpyrocarbonate)-treated water by adding 0.1 ml DEPC per 100 ml distilled water. Stir for at least two hours and autoclave for 30-40 minutes to obtain RNAse-free sterile water.
- For each N or L solution, begin by adding 12.11 g of Tris base to 600 ml of water and stir to dissolve.
- Add KCl to the required final concentration for each buffer as follows:
N1 or L1 29.82 g
N2 or L2 67.10 g
L3 74.55 g
N3 85.73 g
N4 96.92 g
N5 or L5 74.55 g
Stir to dissolve and autoclave.
- Allow the autoclaved buffers to cool to room temperature and add 150 ml of absolute (100%) ethanol to each. (See NOTE)
- Adjust the pH of each buffer with phosphoric acid1 as follows:
N1 or L1 to 6.3
N2 or L2 to 6.3
N3 or L3 to 6.3
N4 to 6.3
N5 or L5 to 8.5
(Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.)
- For each buffer add enough autoclaved, distilled water (DEPC-treated, if required) to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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