This included screening on MDE gels of all the amplicons that did not contain indel polymorphisms from the agarose gel screening.
Briefly, PAD2 (1 μM final) was preincubated with inhibitor (10 or 100 μM final) in PAD2 Gel Screening Buffer (50 mM HEPES pH 7.6, 150 mM NaCl, 1 mM TCEP, 125 μM or 10 mM CaCl2, 0.01% pluronic acid) for 20 min. RFA (5 μM final) was then added, and the reaction was incubated at 37 °C.
Microsatellites with single copy amplicons based on agarose gel screens were assayed for allelic polymorphisms on 6% denaturing polyacrylamide gels using the GenePrint® STR System (Promega).
Full details of the BRCA1 and BRCA2 mutation testing have been reported elsewhere and included: sequencing, protein truncation testing, two-dimensional gel electrophoresis, screening for large genomic alterations, testing for Ashkenazi founder mutations and testing for duplication of exon 13 of BRCA1 [ 3, 12].
Finally, for the remaining few colonies for which the b band still was not pronounced on the secondary gel, we screened individual extracts from an additional 8 10 workers per colony.
Each individual RAGEP marker gel was screened and a similarity matrix was generated.
The gel was screened by using Cy3 excitation for fluorescence detection and an FLA-7000 scanner (Fujifilm, Düsseldorf, Germany).
Additionally, the inhibitory effect of bacteriocins against bacteria was also performed using gel-screening assay.
Alternatively, in order to detect the molecular mass of the bacteriocins, we carried out gel-screening assays using Raoultella sp. and S. agnetis as reporter bacteria.
Then the dried gel was pulverized, since the sieve size of the particle gels were screened with the mesh of 80 (Tyler measurement, opening size: 0.177 mm).
Furthermore, in 2011, the same team reported various advantages of the HRM analysis, and confirmed that this method is much faster than Conformation Sensitive Gel Electrophoresis (CSGE) screening [ 4].
