DNA Ligation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer	The reaction should be performed at 16℃. If good results are not obtained, the reaction time can be extended overnight.

Upstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer
Protocol tips
The reaction should be performed at 16℃. If good results are not obtained, the reaction time can be extended overnight.

Publication protocol

The plasmid encoding FLAG-tag E, C, PrM, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 cDNA clone in pcDNA3.1 (+) flanked by Kpn I ribozyme and Xba I ribozyme sequences as described previously (70). The AA residues at R202W, R226G R464Q of NS3 in the SA strain were swapped individually or both by overlap extension PCR as described previously. Briefly, in a PCR reaction 1 μl of full-length NS3 gene cDNA was mutated and amplified with 20 μM each of 202, 226 or 464 site mutation forward and reverse primers and 20 μM each of JEV-NS3 forward and reverse primers using PrimeSTAR GXL DNA Polymerase (TAKARA) according to the manufacturer’s instructions. The size of PCR products was 1.86 kb and the products were purified by gel purification kit. The PCR mixture was heated at 94 °C for 2 min, followed by 35 cycles of amplification at 98 °C for 10 s, 55 °C for 30s and 68 °C for 1min45s, and a final extension at 68 °C for 10 min. All NS3 fragments with FLAG-tag that included AA mutations and NS3 cDNA vector were digested with enzyme sets KpnI and XbaI (Thermo Scientific). Following the digestions, the NS3 fragments with FLAG-tag and NS3 cDNA vector were ligated together at an approximate molar ratio of 1:3 using TaKaRa DNA Ligation Kit LONG (TAKARA) according to the manufacturer’s instructions.

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Manufacturer protocol

Download the product protocol from Clontech for DNA Ligation Kit below.

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