Diagnostics and therapeutics for restenosis
a technology of restnosis and diagnostics, applied in the field of restnosis diagnostics and therapeutics, can solve the problems of limited long-term success of ptca, extension of local injury or prolongation of the cycle of injury-inflammation-healing, and limited utility of methods for heritable diseases
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example 5
5.5 Example 5
[0305] In this example, the preparation of template DNA is described. PCR-based genotyping does not require particularly high-MW DNA (<20 Kb DNA is often an excellent template). As 100 ng genomic DNA is more than sufficient for single-copy gene amplification, direct amplification from dried blood spots or cell lysates can be used for genotyping, and two of the protocols that we have used are here described below.
[0306] However, if DNA banks need to be established for population studies where DNA needs to be stored for future reference or genotyping at different loci, or where genomic Southern blotting might be needed, good quality high-MW genomic DNA needs to be extracted. Basic buffers and the composition of chemical solutions can be found in major protocol textbooks (Sambrook et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Press; Ausubel and Frederick (1994) Current protocols in molecular biology, John Wiley and Sons).
[0307] Sample DNA can als...
example 6
5.6 Example 6
[0311] In this example, the conditions for conducting appropriate polymerase chain reactions on the collected samples are described. Oligonucleotide primers designed to amplify the relevant region of the gene spanning the polymorphic site (as detailed below) are synthesised, resuspended in Tris-HCl-EDTA buffer (TE) and stored at -20.degree. C. as stock solutions of 200 .mu.M. Aliquots of working solutions (1:1 mixture of forward and reverse, 20 .mu.M of each in water) are prepared in advance of the experiment. Typically PCR reaction mixtures are prepared as detailed below. Divergence from the scheme below can be made for each specific protocol.
18 Stock Final Concentration Volume Concentration Sterile H.sub.20 29.5 .mu.l 10 .times. PCR buffer 200 mM Tris-HCl 5.00 .mu.l 20 mM Tris-HCl, (pH 8.4), 50 mM KCl 500 mM KCl MgCl.sub.2 50 mM 1.75 .mu.l 1.75 mM dNTP mix 10 mM of 4.00 .mu.l 0.2 mM of each each primer forward 20 .mu.M 2.5 .mu.l 1 .mu.M primer reverse 20 .mu.M 2.5 .mu...
example 7
5.7 Example 7
[0314] In this example, the conditions for conducting appropriate polymerase chain reactions on the collected samples are described. A master mix of restriction enzyme buffer and enzyme is prepared and aliquotted in suitable volumes in fresh microwells. We use a multichannel pipette to transfer and mix 25-30 .mu.l of PCR product in the microwells. Digestion is carried out with an oil overlay or capped microtubes at the appropriate temperature for the enzyme on a dry block. Restriction buffer dilutions are calculated on the whole reaction volume (i.e. ignoring salt concentrations of PCR buffer). Restriction enzymes are used 3-5 times in excess of the recommended concentration, to compensate for the unfavorable buffer conditions and to ensure complete digestion.
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