Gfxtm pcr dna and gel band purification kit

In order to study Tngfp insertion sites LMPCR was used according to the protocol described by Prod’hom et al. (1998) (link). This technique allowed the amplification of both ends of the transposon. Briefly, the DNA was digested with the SalI enzyme, the fragments of DNA were ligated with an adapter Sal, containing Salgd and Salpt sequences (Table 2A), and the resulting template was then digested with SalI. The PCR was performed using IS2 and gfp1 primers (specific for Tngfp and directed outward) and the common linker primer Salgd (Table 2A). DNA was denatured by incubating the mixture at 95°C for 5 min. Amplification was achieved using 35 cycles of PCR (95°C for 30 s, specific annealing temperature for 30 s and 72°C for 90 s), followed by a final extension at 72°C for 10 m. Amplified products were separated by standard horizontal gel electrophoresis in a 1.5% agarose gel in TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA) and were stained using ethidium bromide. PCR products were purified using the GFX^TM PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech Inc.) followed by ExoSAP-IT^® PCR product clean up (Affymetrix).
The amplified products were sequenced with the corresponding oligonucleotides using CNIO³ service and further checked for homology at Tuberculist⁴, Bovilist⁵ and NCBI⁶ database BLAST analysis.

We used two molecular markers in the study: ITS (ITS-1, 5.8S, and ITS-2) and atpB-rbcL. For the putative new species, we extracted total genomic DNA from silica gel-dried leaves using a CTAB protocol modified from that of Doyle and Doyle (1987) (link). For the primers and PCR protocols for ITS and atpB-rbcL, we followed White et al. (1990) (link) and Taberlet et al. (1991) (link), respectively. Subsequently, we purified the PCR products using a GFX^TMPCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and sequenced the markers using an ABI Prism Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), while analyzing the PCR products on an ABI3730xl automated DNA sequencer.

The DNA sample was amplified by polymerase chain reaction (PCR) using TaKaRa LA Taq^TM (Takara Bio Inc., Kusatsu, Japan) with GC buffer II, included in the kit. The forward primer was 20 pmol/μL of ITS5 and the reverse primer was 20 pmol/μL of ITS4 [45 ]. The samples were incubated for an initial 2 min at 94°C and then 33 cycles of 50 s denaturation at 94°C, 1 min annealing at 48°C and 30 s extension at 72°C. When the amplification was insufficient, 20 pmol/μL of AB101 for forward and AB102 primers for reverse [46 (link)] were used instead of ITS5 and ITS4 primers. The samples were incubated for an initial 2 min at 94°C and then 33 cycles of 50 s denaturation at 94°C, 1 min annealing at 60°C, and 30 s extension at 72°C. The PCR products were then purified from collected agarose gels containing the targeted DNA region using the GFX^TM PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Piscataway, New Jersey, USA) following the manufacturer’s protocol. For cycle sequencing, the samples were incubated for an initial 1 min at 96°C, and then 35 cycles of 10 s denaturation at 96°C, 5 s annealing at 50°C, and 80 s extension at 72°C.