CpoI (RsrII) (10 U/µL)

Protocol tips

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	All purified PCR products were double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific) and purified using a QIAquick PCR purification kit (Qiagen). Vector pBacT7-S1T3D (plasmid 33282; Addgene) was double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific), and vector backbone (5,716-bp fragment) was gel extracted using a PureLink Quick Gel Extraction kit (Fisher Scientific).

Protocol tips

All purified PCR products were double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific) and purified using a QIAquick PCR purification kit (Qiagen). Vector pBacT7-S1T3D (plasmid 33282; Addgene) was double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific), and vector backbone (5,716-bp fragment) was gel extracted using a PureLink Quick Gel Extraction kit (Fisher Scientific).

Publication protocol

"L929 cells in six-well plates were infected with T3DPL or T3DTD at an MOI of 3, and cell lysates were collected at 24 h postinfection (hpi) in TRI reagent (Millipore Sigma). Total RNA was purified using as per the TRI reagent protocol. Using 1 μg of RNA per 20-μl reaction volume, cDNA synthesis was performed with pooled forward and reverse gene-specific primers for all 10 genes and with Moloney murine leukemia virus (M-MLV) reverse transcriptase (ThermoFisher Scientific) as per the M-MLV reverse transcriptase protocol. Using cDNA (2 μl) as a template, each gene segment was PCR amplified (100-μl total reaction volume) using a gene-specific primer set with an iProof High Fidelity PCR kit (Bio-Rad), as per the manufacturer’s protocol. PCR products were purified using a QIAquick PCR purification kit (Qiagen). S1, M1, M2, M3, L1, L2, and L3 gene segments were gel extracted using a PureLink Quick Gel Extraction kit (Fisher Scientific). All purified PCR products were double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific) and purified using a QIAquick PCR purification kit (Qiagen). Vector pBacT7-S1T3D (plasmid 33282; Addgene) was double digested with CpoI (ThermoFisher Scientific) and NotI (ThermoFisher Scientific), and vector backbone (5,716-bp fragment) was gel extracted using a PureLink Quick Gel Extraction kit (Fisher Scientific). Gel-extracted vector backbone was treated using alkaline phosphatase (ThermoFisher Scientific) as per the manufacturer’s protocol and purified using a QIAquick PCR purification kit (Qiagen).

Ligation reactions were performed with a 1:5 ratio of vector/insert using T4 DNA ligase (ThermoFisher Scientific) in a 20-μl reaction volume as per the manufacturer’s protocol. Ligation reactions were transformed into Stbl3 chemically competent bacteria, and transformants were selected on LB-agar-carbenicillin (100 μg/ml) plates. For each gene, 8 bacterial colonies were selected for colony PCR validation. Positive bacteria clones were inoculated into 50 ml of LB-carbenicillin (100 μg/ml) culture for amplification and plasmid purification using a GenElute HP Plasmid Midiprep kit (Millipore Sigma). All plasmids except the plasmid expressing T3D-L1 were amplified at 37°C for 14 to 16 h. T3D-L1 plasmids were amplified at 30°C for 24 to 28 h. All plasmids were sequenced to validate inserted gene sequences."

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for CpoI (RsrII) (10 U/µL) below.

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