KpnI-HF®

Protocol tips

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	The 1.7-kb and 1.5-kb PCR products for HA and NP, respectively, were purified from agarose gel slices using Qiagen gel purification kit and digested with NheI and KpnI in a 40-μl reaction that contains 1× CutSmart Buffer, 1 μl of NheI (10 Units), and 1 μl of KpnI-HF (20 Units), at 37°C for 4 h (See Note 1). The digested PCR fragments were purified using Qiagen nucleotide removal kit.

Protocol tips
The 1.7-kb and 1.5-kb PCR products for HA and NP, respectively, were purified from agarose gel slices using Qiagen gel purification kit and digested with NheI and KpnI in a 40-μl reaction that contains 1× CutSmart Buffer, 1 μl of NheI (10 Units), and 1 μl of KpnI-HF (20 Units), at 37°C for 4 h (See Note 1). The digested PCR fragments were purified using Qiagen nucleotide removal kit.

Publication protocol

"PCR amplification of antigen genes: Influenza A virus HA and NP genes are used as model antigens to explain the construction and usage of the rP18tri-based vaccine candidates. HA and NP open-reading-frames (ORFs) were amplified from the pPolI-HA and pPolI-NP plasmids, respectively, with the 5’ primers containing the NheI site (HA Forward and NP Forward) and the 3’ primers containing the KpnI site (HA Reverse and NP Reverse), in a 50-μl reaction that contains 1× Phusion HF buffer, 50 ng of plasmid DNA, 25 pmol of each primer, 10 uM dNTPs, and 1 unit of Phusion DNA polymerase. The PCR conditions were 98°C for 30 sec, and 30 cycles of 98°C for 10 sec, 55°C for 15 sec, and 72°C for 30 sec. The 1.7-kb and 1.5-kb PCR products for HA and NP, respectively, were purified from agarose gel slices using Qiagen gel purification kit and digested with NheI and KpnI in a 40-μl reaction that contains 1× CutSmart Buffer, 1 μl of NheI (10 Units), and 1 μl of KpnI-HF (20 Units), at 37°C for 4 h (See Note 1). The digested PCR fragments were purified using Qiagen nucleotide removal kit.

Digestion of the viral vector plasmids. One microgram of each of the pP18S1-GPC/MCS and pP18S2-MCS/NP plasmids (Fig. 2) was digested with NheI and KpnI in a 30-μl reaction that contains 1× CutSmart Buffer, 1 μl of NheI (10 Units) and 1 μl of KpnI-HF (20 units), at 37°C for 4 h (See Note 1). At the end of the restriction enzyme reaction, 3 μl of 10× Antarctic Phosphatase Buffer and 1 μl of Antarctic Phosphatase were added and incubated at 37°C for 1 h followed by a 5-min incubation at 70°C to inactivate the enzyme.

Ligation and transformation. The NheI/KpnI-treated HA PCR fragment was ligated into the digested pP18S1-GPC/MCS vector, while the NheI/KpnI-treated NP PCR fragment was ligated with the digested pP18S2-MCS/NP vector, at an insert-to-vector ratio of 3 to 1. The 10-μl ligation reaction contains 1 μl of T4 ligase buffer, 1 μl of 10mM dNTPs, and 1μl of T4 DNA ligase (400 Units) (See Note 2). After incubating at room temperature for 2 h (See Note 3), 5 μl of the ligation reaction was used to transform 100-μl of E. coli competent Stbl2 cells that were plated on LB-Amp plate (See Note 4). Plasmid DNAs were extracted from the bacterial cultures using the Qiagen mini-prep kit and screened by restriction enzyme digestion using the NheI and KpnI-HF enzymes. The positive clones were verified by sequencing and named pP18S1-GPC/H and pP18S2-P/NP, respectively."

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