Abi 3130 l genetic analyzer

The MLPA reaction is an efficient and reliable technique for the analysis of exon copy numbers. We designed a pair of MLPA probes for each HMGCL exon, using the human MLPA probe design program (H-MAPD), as previously described (12 (link)). The MLPA probe sets for the HMGCL exon are listed in Table II. MLPA reactions were performed according to the manufacturer’s instructions (MRC-Holland BV, Amsterdam, The Netherlands) using 100 ng of genomic DNA, the EK1 MLPA reagent kit and the P200-A1 Human DNA reference kit, which includes reference probes and MLPA control fragments (MRC-Holland BV). The PCR products were separated by capillary electrophoresis on an ABI 3130×l genetic analyzer (Applied Biosystems, Warrington, UK). GeneMapper v4.0 software (Applied Biosystems) was used to analyze the separated products and to retrieve peak intensities corresponding to each probe in the different samples. Integrated peak areas were exported to an Excel 2003 spreadsheet. Data generated from a combination of the HMGCL synthetic probe mix and the P200-A1 probe mix were intra-normalized by dividing the peak area of the amplification product of each probe by the total area of only the reference probes in P200-A1. Secondly, normalization was achieved by dividing this intra-normalized probe ratio in a sample by the average intra-normalized probe ratio of all reference samples.

The binding of RctB was performed under the same condition as was used for EMSA. The binding reactions were treated with 0.01 unit of DNase I (Promega) for 1 min at room temperature. DNaseI was inactivated by adding 15 mM EDTA followed by heating (95°C, 10 min). The DNA was purified using Qiaquick PCR purification kit (Qiagen). Primer extension was performed using 6 FAM-labeled primer and Thermo Sequenase polymerase (USB) according to the manufacturer's protocol. Amplified products were purified using Qiaquick PCR purification kit. The purified products along with GeneScan 500-ROX size standard (Applied Biosystems) were denatured by adding Hi-Di formamide (Applied Biosystems) followed by heating (75°C, 10 min) and immediately chilled on ice. The single stranded DNA was analyzed on an ABI 3130×l Genetic Analyzer (Applied Biosystems) and data was analyzed using GeneMapper (v. 3.7) software.

The total DNA was extracted from gill lamellae using E.Z.N.A E-Z 96 Tissue DNA Kit (Omega-Biotek) following the manufacturer’s protocol. Individuals were genotyped at 20 polymorphic microsatellite loci: BFRO-018 [60] (link), BWF1, BWF2 [61] , Cla-Tet01, Cla-Tet03, Cla-Tet06, Cla-Tet09, Cla-Tet10, Cla-Tet13, Cla-Tet15, Cla-Tet17, Cla-Tet18 [62] (link), Cocl-Lav04, Cocl-Lav06, Cocl-Lav10, Cocl-Lav18, Cocl-Lav27, Cocl-Lav49, Cocl-Lav52 [63] , C2-157 [64] (link). The loci were co-amplified in four 2.5 µl PCR multiplex reactions as described earlier [65] using the QIAGEN Multiplex PCR kit following the manufacturer’s protocol. The PCR products were denatured in Hi-Di Formamide, containing LIZ-500 internal size standard (Applied Biosystems) and separated using an ABI-3130×l Genetic Analyzer (Applied Biosystems). The alleles were scored using the automatic binning function, with predefined bins, as implemented in Gene-Mapper 3.7 software (Applied Biosystems). All alleles were subsequently verified visually by two independent persons. In addition, the included replicate and blank samples were manually verified to ensure the validity of the data. Finally, we manually verified all identified private alleles to ensure that they are not an artefact from inconsistent scoring.

Archaeal and bacterial 16S rRNA gene fragments for T-RFLP analysis were amplified by PCR using the primer pairs 5′ FAM-labeled Arch21F/Ar912r and 5′ FAM-labeled EUB338F/1492R, respectively. PCR was performed under the conditions described above. The PCR products were digested with HaeIII and HhaI (TaKaRa Bio Inc., Otsu, Japan) separately. The labeled fragments were analyzed by electrophoresis on an ABI 3130×l Genetic Analyzer (Applied Biosystems, CA, USA). GeneScan 1200 LIZ (Applied Biosystems) was used as a size standard.

All PCR amplifications were performed as described (Roche protocol for Faststart Taq DNA polymerase). The following PCR primers were used (i) TNFRSF13B sense: 5′-TACTTGGCTTACTCTGGAAT-3′ and anti-sense: 5′-CATTTGCTTGGACTCTGG-3′ and (ii) TCF3 sense: 5′-TCTCTTGACCTCGTGATCTG-3′, anti-sense 5′-GACTCACCGAGGATGGAA-3′.
DNA sequencing was performed with Big Dye Terminator cycle sequencing on an ABI 3130 × l Genetic Analyzer according to the manufacturer’s standard protocol and reagents (Applied Biosystems, Waltham, MA, USA). Sequence electropherograms were compared with wild-type sequences using SeqMan v5.01 software (DNASTAR, Madison, WI, USA).