Pgem t easy vector system 2 kit

Sequencing of SGPL1 transcript variants was done by Seqlab Sequencing Laboratories (Göttingen, Germany). Forward and reverse sequencing was performed after amplification of SGPL1 coding sequence with the forward primer fw: 5′-ATGCCTAGCACAGACCTTCT-3′ and reverse primer: 5′-CTTCCTGGTGAGCTTAAAACA-3′ to determine alterations in the SGPL1 sequence. For determination whether the SGPL1 mutation in the coding sequence is homozygous or heterozygous, the purified SGPL1 PCR fragments generated by a thermostable Taq polymerase with proofreading function were cloned in the pGEM-T Vector via TA-cloning procedure using the pGEM®-T Easy Vector System II Kit (Promega Corp., Madison, USA) according to the manufacturer’s instructions. Briefly, the purified SGPL1 PCR fragments were A-tailed through dATP and Taq polymerase incubation for 15 min at 70 °C and further ligated into the linearized T-tailed pGEM®-T Easy Vector for 1 h at 24 °C. Afterwards the ligation product was transformed into JM109 competent E.coli cells. The competent cells were plated on LB/amp/IPTG/X-gal plates and the recombinant cells were identified by blue/white screening on indicator plates. Finally, ten SGPL1 recombinant clones were sequenced.

1 pg to 50 ng of the total community DNA was amplified in a 50 μl reaction mixture containing 5 μl of 10 × PCR buffer (MBI, Fermentas), 45 pmol of each primer, 0.2 mM of each of the four dNTPs and 2.5 U of Taq polymerase (MBI, Fermentas) using bacteria-specific forward primer 5′-AGA GTT TGA ACA TGG CTG-3′ (S-D-Bact-0027-a-S-18) and reverse primer 5′-CTA GCG ATT CCG ACT TCA-3′ ( S-D-Bact-1327-a-A-18) [36 (link)]. The numbers refer to the positions in the Escherichia coli 16S rRNA [37 (link)]. Reaction mixtures were incubated in a thermal cycler (GeneAmp 2400 PCR system, PE Applied Biosystems) for an initial denaturation at 94°C for 2 min followed by 40 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 2 min. Amplified DNAs were gel purified (QIAGEN Gel Extraction kit, QIAGEN, Germany). The purified DNAs were cloned directly by the TA cloning method (30) with a pGEM-T easy vector system II kit (Promega). The clones were sequenced using vector specific primers in an ABI Prism 3100 (16 capillary) sequencer (Applied Biosystems) according to manufacturer's instruction.

RT was carried out with Super Script III Reverse Transcriptase (Thermo Fisher Scientific). Initial pre incubation at 65 °C for 5 min with 0.5 mM of each dNTP, 0.5 μM Primer mir5 and 11 μL RNA were carried out. In a second step a mix was prepared with 1X First-Strand Buffer, 5 mM DTT, 0.2 U RNaseOUT and 10 U SuperScript III RT in a final volume of 20 μL, the mix was incubated at 55 °C for 90 min, and 70 °C for 15 min.
PCR amplifications were performed using Expand High Fidelity Plus PCR System (Roche Diagnostics, Basel, Switzerland). Briefly, 0.05 U Expand High Fidelity Plus Enzyme Blend, 1X Expand High Fidelity Plus Reaction Buffer with 1.5 mM MgCl₂, 0.2 μM Primers A5 and B3 (included multiplex identifiers at the 5′ end), 0.2 μM of each dNTP, 4 μL cDNA and RNAse free water to a final volume of 50 μL. PCR conditions were 94 °C for 3 min, 20–25 cycles at 94 °C for 30 s, 57 °C for 45 s and 72 °C for 1 min followed by 71 °C for 7 min. The number of cycles was optimized for each library to avoid saturation. The resulting PCR was purified with the QIAquick PCR purification kit (Quiagen, Hilden, Germany).
Cloning in the pGEM-T Easy Vector System II Kit (Promega Corporation, Wisconsin, United States) and posterior Sanger sequencing with the ABI-PRISM 3700 (Thermo Fisher Scientific) were carried out to check the libraries construction prior to the HTS.