Agarose gel dna purification kit

The 2^nd–11^th fractions of each sample (10 samples; total 100 fractions) were analyzed with T-RFLP fingerprinting as described by Zhang et al. [33 (link)]. Briefly, the fluorescently labeled forward primer 27F (5' [6FAM]-AGAGTTTGATCMTGGCTCAG-3') and the unlabeled reverse primer 927R (5'-ACCGCTTGTGCGGGCCC-3') were used to amplify bacterial 16S rRNA genes [34 , 35 (link)]. PCR products were checked by electrophoresis on 1% agarose gel and purified with an agarose gel DNA purification kit (Tiangen Biotech Co., Ltd., Beijing, China). Purified products were digested with restriction enzyme FastDigest RsaI (Thermo Fisher Scientific, Inc.) at 37°C for 1–2 h. The digested products were recovered using 20 uL of sterile deionized water and ethanol precipitation. Purified products were then mixed with 0.5 uL of an internal size standard (ET ROX-900) and then detected using a MegaBACE genetic analyzer (Amersham Biosciences Corp., Piscataway, NJ, USA). The output was transferred to T-REX software (http://trex.biohpc.org/) [33 (link), 36 (link)] for noise removal and construction of a data matrix. The obtained matrix was further analyzed with Primer 5 analysis software to determine fragment profiles of the 12 density gradient fractions from each experimental sample as well as community similarity between fractions. Typical ¹³C- and ¹²C-fractions were chosen for pyrosequencing.

E. coli strains JM109 and BL21(DE3) were used as a cloning vehicle and an expression host, respectively. The EZ-10Spin Column Plasmid Mini-Prep kit and agarose gel DNA purification kit were purchased from Tiangen Co. Ltd (Beijing, China). The vectors pMD^™18-T and pET-24a(+) were purchased from Novagen (Shanghai, China). The PrimeSTAR^®HS DNA Polymerase, restriction enzymes and T4 DNA ligase were obtained from Takara (Dalian, China). Restriction endonuclease DpnI was obtained from Generay Biotech (Shanghai, China). GABA, PLP, and 1,2-phthalic dicarboxaldehyde were obtained from Sigma Chemical Co. Ltd. (Shanghai, China). Other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd. (SCRC, Shanghai, China).

First-strand cDNA synthesis was accomplished using the SMARTer RACE 5′/3′ Kit (Takara, Dalian, China) according to the manufacturer’s protocol. 5′ RACE and 3′ RACE reactions were performed by nested PCR, using the SLC35D3-specific primers GSP5/3 and NGSP5 and the universal primers UPM long and UPM short (Table 1).
All PCR products, including the internal fragment and fragments generated by 5′ RACE and 3′ RACE, were subjected to agarose gel electrophoresis, then recovered using an agarose gel DNA Purification Kit (Tiangen, Beijing, China). The products were cloned into the pEASY-T1 vector (Trans, Beijing, China). Clones were submitted to Sangon Biotech Co., Ltd. (Shanghai, China) for nucleotide sequencing.