DNA was denatured at 95 °C for 10 min prior blotting on to Hybond N+ (Amersham Pharmacia Biotech, England) membrane in a slot blot apparatus (Cleaver Scientific Co., England) under vacuum. The membrane was UV cross-linked (Church and Gilbert 1984) at 12 × 104 μJ/cm2 energy in a CL-1000 Ultraviolet crosslinker (UVP, Upland, CA, USA).
Illustration of a human DNA quantitation result with the slot blot procedure. A serial dilution of a human DNA standard is run on either side of the slot blot membrane for comparison purposes. The quantity of each of the unknown samples is estimated by visual comparison to the calibration standards. This is a simple slot/dot blot protocol for detecting antigen on a membrane. Using the method outline here, one can achieve results in under 90 min because it eliminates the need. Load genomic DNA probes along with the marker (e.g. DNA Markers for Genomic DNA analysis) on 0.7% agarose gel (20 cm length). Run for 18 hours at 3 V/cm in 1X TAE buffer. Abstract: This protocol describes how to prepare a DNA dot blot with a dot blot apparatus and a vacuum manifold. Author: Simon Dawson.
Often it is informative to quantify the abundance of a certain RNA or DNA in the extracted nucleic acid mixture by dot blot or slot blot hybridization without prior digestion and electrophoresis. In the procedure, the nucleic acid mixture is blotted to a membrane where the hybridization is carried out. The difference between dot and slot blot procedures is in the way that the nucleic acid mixture is blotted onto the membrane. In dot blotting the nucleic acids are blotted as circular blots, whereas in slot blotting they are blotted into rectangular slots (Figure 1). The latter method allows a more precise observation of different hybridization signal intensities. Quantification of a certain RNA/DNA compared with total RNA/DNA can be obtained by hybridization with universal and specific oligonucleotide probes. The relative abundance is calculated by dividing the amount of specific probe bound to a given sample by the amount of hybridized universal probe measured e.g. as fluorescence intensity (fluorescent probes) or counts per minute (radioactively labelled probes).
Figure 1. Dots and slots in dot and slot blot hybridization, respectively.
Dna Slot Blot Protocol Igg
One should be aware that the data of relative DNA or RNA abundance can not be directly translated into cell numbers. Cells may have different copy numbers of various genes. Moreover, cells of different species have different ribosome contents ranging roughly between 103 and 105 ribosomes per cell. Even for one strain, cellular rRNA contents can vary significantly (at least over one order of magnitude), since they are directly correlated with the growth rate. The relative rRNA abundance should, however, represent a reasonable measurement of the relative physiological activity of the respective population, since it is the product of the number of detected cells and the average rRNA content. This information on the general activity of a given population should not automatically be regarded as an indication of a specific kind of activity. Often, one population has the potential to catalyze different transformations - one genotype is linked to several phenotypes.