Abstract
Purification of the probe is essential as nonspecifically labeled molecules increase the background on the hybridized filters. The purification of the restriction-digested primed-extended DNA is performed on a preparative denaturing polyacrylamide gel. The probe can be eluted from the polyacry- lamide gel either by diffusion or by electroelution. Of all the different pro-cedures we have tested the worst recovery was obtained by the diffusion method (50% recovery). The recoveiy by electroelution was usually much better (70–95%). Electroelution was carried out using the Sartorius-col- lodium bags or in an IS CO-apparatus as described in the following ‘Step-by- Step Procedure’. The recoveries range from 70 to 90% in both systems. Nevertheless, the use of bad batches of collodium membranes can result in low recovery of probe. After the electroelution, the eluate has to be filtered (Millex filters) to give a cleaner result in the hybridization. If the probe is not filtered, the background on the hybridized nylon membranes can be very high and may have a speckled appearance on autoradiography. Since the volume of the filtered eluate is too large for the direct hybridization (see ‘Step-by-Step Procedure’) it must be ethanol precipitated.KeywordsSaran WrapPhenol BlueCollodium MembraneSpeckled AppearanceISCO SampleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Published Version
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