Dot/Slot blotting is a technique where (un-fractionated) nucleic acids or protein are immobilized (blotted) on to the membrane in dot or slot geometries, often with the intention of comparative hybridization analysis. This enables studying the abundance of a target molecule in a large number of samples blotted at one time. Dot and slot blots are typically prepared with the aid of a manifold and suction device. The use of such an apparatus makes spotting faster and more reproducible than manual blotting and is the preferred method if many blots are to be prepared at any one time.
We have Bio-Rad’s Bio-Dot Microfiltration Apparatus, however the Bio-Dot SF apparatus is a better choice as the Bio-Dot SF applies samples in a thin line instead of a circle which enables easy densitometeric quantification. The Bio-Dot can be converted to Bio-Dot SF by acquiring Bio-Dot SF module.
The components of the Bio-Dot Microfiltration Apparatus are 1) Sealing gasket which prevents cross contamination of samples between the wells 2) Sample template that enables easy sample application particularly with multi channel pipettes 3) Gasket support plate 4) Vacuum manifold 5) Three-way flow valve that allows easy vacuum adjustments from passive filtration to rapid vacuum-assisted washes. The apparatus is autoclavable and is resistant to many chemicals (including acids, bases, and ethanol); it can be treated with 0.4 M NaOH solution or DEPC water and autoclaved, allowing for work in nuclease-free conditions.
Briefly, the apparatus is cleaned and assembled by positioning the sealing gasket and gasket support plate over the vacuum manifold. There is only one way that the gasket support plate can slide into the manifold. The activated nitrocellulose membrane is then laid over the gasket support, taking care to avoid air bubbles. The sample template is then laid on top of the membrane. After the four screws are finger-tightened in a diagonal manner, a vacuum source is attached to the flow valve with a waste trap set up and positioned between the vacuum outlet and flow valve. The vacuum is turned on and the valve is set to 3-way valve to apply vacuum to the apparatus. The flow valve is adjusted so that the vacuum manifold is open to air after which 100 µl of buffer is applied to all of the sample wells. The buffer is then removed from the wells by vacuum and then the vacuum is turned off. This allows rehydration of the membrane. Now the samples are loaded; sample volume should be at least 200 µl. DNA and RNA samples have to be denatured before spotting; denature by adding NaOH and EDTA solutions to final concentrations of 0.4 M and 10 mM, respectively, and heating the sample to 100°C for 10 minutes.
My lab focus is to understand molecular alterations during sequential hepato-carcinogenesis. To study amplification status as well as transcription rate of few sets of genes during hepato-carcinogenesis, we perform dot blot and run-on transcription assays. As plenty of samples and genes are to be studied, it becomes convenient to spot genomic DNA or cDNA with the Bio-Dot Microfiltration Apparatus.
In conclusion, the Bio-Dot Microfiltration Apparatus saves loads of time and yields reproducible results.
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Nidhi Vishnoi
Senior Research Fellow
Chemical Carcinogenesis
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), TMC
India
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