3. Reaction mix preparation: Mix enough reagents for the number of assays performed.
The PCR mix preparation and the HRMA procedure were carried out with the identical methodology as described in the MS-HRMA of DLC-1 section.
DNA sequences for the PMP22 probe set and for other MAPH probes along with protocols for probe set and probe mix preparation can be found at [ 27].
DNA extraction was performed in the BSL3 laboratory, master mix preparation in a second room, and PCR and hybridisation were performed in a third laboratory.
In order to prevent assay contamination, sample processing (DNA-extraction, template preparation, centrifugation, aliquoting, and master mix preparation) and PCR-amplification were performed in dedicated laboratory facilities certified for molecular diagnostics and especially for PCR, using standard precautions.
8-methoxysporalen was added during the mix preparation to intercalate between double-stranded nucleic acids generated during amplification, thereby limiting carry-over contamination after UV irradiation and before PCR product analysis.
2. Sample preparations: Prepare test samples in 80 μl/well with reaction buffer in a 96-well plate, include UDP-GlcNAc (0.5 mM), peptide (0.5 mM), and varying amount of OGT. 3. Reaction mix preparation: Mix enough reagents for the number of assays performed.
PCR Mix preparation: A 50 μl PCR reaction was performed containing 3μl of DNA, 10X PCR buffer (Fermentas, USA), 1.5 mM MgCl2, 300 M each of dATP, dGTP, dCTP and dTTP, 1.5 U Taq DNA polymerase (Fermentas, USA) and 25 pM each of forward and reverse primers.
Otherwise, go directly to mix preparation.
A10 Mixed Preparation Results Federal workers across Washington are preparing for possible terrorist attacks, but officials are fumbling over readying their agencies and the results have varied widely.
