High Pure PCR Product Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.	Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.
Protocol tips
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Publication protocol

Polymerase chain reaction amplification of SOmp2b
Chromosomal DNA of strain 544 (Pasteur Institute of Iran) was prepared according to the manufacturer's protocol of DNA extraction kit (Roche, Mannheim, Germany). Polymerase chain reaction (PCR) was performed using forward 5'-CTAATGGCCGACG CAATCGTCGC-3' and reverse 5'-CCGTTAGAACGAACGCTGGAAGCG-3' primers with I and I restriction sites (underlined), respectively. The amplification was carried out in a 50 µL total volume component of 500 ng template DNA, 1 µM each primer, 200 µM dNTP-mix, 1× pfu buffer containing MgSO, and 1 unit of pfu DNA polymerase. PCR conditions used for gene amplification were as follows: hot start at 95℃ for 3 minutes, followed by 30 cycles of denaturation at 94℃ for 45 seconds, annealing at 55℃ for 45 seconds and extension at 72℃ for 1 minute and a final extension at 72℃ for 7 minutes. High pure PCR product purification kit (Roche) was used for purification of the PCR products.

Cloning of SOmp2b gene in pET28a expression vector
The purified SOmp2b PCR product and pET28a expression vector (Novagen, Madison, WI, USA) were digested with I and I (Thermo Scientific, Waltahm, MA, USA) restriction enzymes. The ligation reaction has been carried out using T4 DNA ligase (Thermo Scientific) at 4℃, overnight. Transformed BL21 (DE3) (Stratagene, Kirkland, WA, USA) competent cells were grown on Luria-Bertani agar containing 50 µg/mL of kanamycin (Gibco, Grand Island, NY, USA). Finally, pET28a-SOmp2b positive clones were identified by clone-PCR, restriction enzyme digestion, and sequencing.

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Manufacturer protocol

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