3730xl dna analyzer platform

Rapid amplification of cDNA ends (RACE) was used to identify and isolate the 5′- and 3′-end of Arabian camel HSPB-1 by using a RACE kits (Invitrogen, Carlsbad, CA, USA). Total RNA was annealed with 5′- and 3′-end primers (Table 1), and reversely transcribed respectively to the respective 5′- and 3′-cDNA. The resulting first-stranded 5′- and 3′-cDNA were then utilized as templates in PCR. The cycling program was set for five cycles of 95°C for 4 min; 5 cycles of 95°C for 15 s, 70°C for 15 s, 72°C for 3 min; 5 cycles of 95°C for 15 s, 68°C for 15 s, 72°C for 3 min; 5 cycles of 95°C for 15 s, 65°C for 15 s, 72°C for 3 min; 25 cycles of 95°C for 15 s, 60°C for 15 s, 72°C for 3 min; 1 cycle of 72°C for 5 min. The purified nested PCR product was ligated into pcDNA5/FRT/TO GFP-tagged vector (a gift from Harm Kampinga; Addgene plasmid No., 19487) [26 (link)] by using BamHI (NEB R3136S) and NotI (NEB R3189S) restriction sites. Subsequently, 5 ul of the ligation mixture was used as a template to transform chemically modified DH5α competent cells (ThermoFisher Scientific). The cloned Arabian camel HSPB-1 was sequenced using Applied Biosystems 3730xl DNA Analyzer platform (Applied Biosystems, Foster City, USA). The conditions of the chain termination PCR were as follows: one cycle at 94°C for 35 s, followed by 25 cycles at 94°C for 40 s, 50°C for 35 s, and 60°C for 1 min.