Spotted hybridization

spot hybridization (Dot blot) is to be examined specimen point to the membrane, baking fixed. This method is time-consuming and can be semi-quantitatively analyzed. Multiple samples can be detected simultaneously on a single membrane, and for the convenience of accurate and convenient point samples, there are a variety of multi-tube suction machines (Manifold) on the market, such as Minifold I. and II., Bio-Dot and Hybri-Dot, which have many holes, and the samples are added to the holes and flow to the membrane under negative pressure in a speck or slit. Repeatedly rinse in the sample hole, remove the membrane roasted dry or ultraviolet radiation to fix the specimen, at this time the membrane can be hybridized.
(1)
DNA
speckled hybridization:
(1) first soaks the membrane in water and then places it in × 15 SSC.
(2) dissolve the DNA sample in water or TE, bring to the boil 5min, and cool the ice at medium speed.
(3) pencil on the membrane to mark the position, DNA dot on the membrane, each sample general point 5 μl (2 to 10 μg DNA).
(4) to dry the film, seal to save spare.
(2) RNA speckled hybridization: Similar to the above method, each sample must be added up to 10 μg of total RNA (purified by phenol/chloroform or isothionyl cyanate extraction) by dissolving RNA in 5 μl
DEPC water, plus 5 μl formaldehyde/SSC buffer (10×SSC containing 6.15mol/L formaldehyde), denatured RNA, and then took 5-8 μl sample on the treated membrane, drying.
(3) Complete cell speckle hybridization: using similar methods to detect bacterial fallout, the specific sequence of cell
cultured
can be quickly detected, the entire cell point to the membrane, by NaOH treatment, so that DNA exposure, degeneration and fixation, and then according to the conventional method of hybridization and detection. This method has been used to detect at least 5pg of Epstein-Barr virus DNA from 105 cultured cells. Complete cell speckle imprinting can be used to screen a large number of specimens, because it allows cells to dissolve directly on the membrane, so the DNA content is even higher than the commonly used extraction method, and does not affect crossbreeding with 32P-labeled probes, but it is not suitable for non-radioactive marker probes, because DNA purity is not enough, will produce a high background.