SalI R6051

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The purified PCR products were then digested with NdeI (Promega) and SalI (Promega) at 37°C for 1 h.	The restriction digests were purified as described above with a Qiagen QIAquick PCR purification kit and eluted in 30 μl ddH2O.

Protocol tips
The purified PCR products were then digested with NdeI (Promega) and SalI (Promega) at 37°C for 1 h.
Downstream tips
The restriction digests were purified as described above with a Qiagen QIAquick PCR purification kit and eluted in 30 μl ddH2O.

Publication protocol

The Sulfolobus overexpression vector pSeSD1 (modified from reference 17) was digested with NdeI and SalI (Promega, Madison, WI) at 37°C for 1 h. The linearized vector was dephosphorylated with calf intestinal alkaline phosphatase (CIAP; Promega) at 37°C for 1 h. The vector was purified using phenol-chloroform-isoamyl alcohol (25:24:1) extraction followed by ethanol precipitation. The ORF c92 was PCR amplified from STIV-TOPO (6, 24) using iProof high-fidelity polymerase (Bio-Rad, Hercules, CA) under standard conditions. The primers were designed to introduce an NdeI restriction endonuclease site on the 5′ end, a FLAG tag, and a SalI restriction endonuclease site on the 3′ end of the gene. The PCR conditions were: 5× HF iProof buffer, 10 mM dNTPs, 50 pmol/μl c92-F-NdeI (5′-GACTCTAACAAAGTATTTTCATATGGTAGCAGAAATAGC-3′; the NdeI site is underlined in the primer sequence), 50pmol/μl c92-R-FLAG+SalI (5′-GCCAAGAAAAAAAAGATAGTCGACTTTATCATCATCATCCTTATAATCCTTTTGAGCGGGCTG-3′; the SalI site is underlined and the FLAG tag sequence is in italics), 1U iProof polymerase, and 1 ng/μl STIV-TOPO DNA in a total volume of 50 μl total. The cycling conditions were as follows: initial denaturation of 98°C for 1 min; 30 cycles of 98°C for 15 s, 61°C for 15 s, and 72°C for 15 s; and a final extension for 10 min at 72°C. The size of the PCR amplicon was verified using agarose gel electrophoresis, and the correct products were purified using a QIAquick PCR purification kit (Qiagen, Germantown, MD) and eluted in 30 μl double-distilled H2O (ddH2O). The purified PCR products were then digested with NdeI (Promega) and SalI (Promega) at 37°C for 1 h. The restriction digests were purified as described above with a Qiagen QIAquick PCR purification kit and eluted in 30 μl ddH2O. The PCR products and the pSeSD1 vector were ligated with T4 DNA ligase (Promega) overnight starting at 16°C and decreasing to 4°C in a water bath. The ligations were transformed into XL2-Blue Ultracompetent cells (Stratagene, Santa Clara, CA). Resulting transformants were screened using a PureYield plasmid miniprep system (Promega) to obtain plasmid DNA. The isolated plasmid DNA was restriction endonuclease mapped by digestion with NdeI and SalI and sequenced to verify the c92+FLAG construct

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Manufacturer protocol

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