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A High-Throughput UAS-cDNA/ORF Plasmid Library Construction Using CRISPRmass
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Large-Scale Transformation of UAS-cDNA/ORF Plasmids from CRISPR-Based Modular Assembly into E. coli
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JoVE Journal Genetik
Large-Scale Transformation of UAS-cDNA/ORF Plasmids from CRISPR-Based Modular Assembly into E. coli

Large-Scale Transformation of UAS-cDNA/ORF Plasmids from CRISPR-Based Modular Assembly into E. coli

A High-Throughput UAS-cDNA/ORF Plasmid Library Construction Using CRISPRmass

DOI:
10.3791/201692-v

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01:46 min

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May 17, 2024

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, , , , , , ,
1Clinical Research Institute, The Affiliated Nanhua Hospital, Hengyang Medical School,University of South China, 2Department of Neurology, The Affiliated Nanhua Hospital, Hengyang Medical School,University of South China, 3Department of Clinical Laboratory, The Affiliated Nanhua Hospital, Hengyang Medical School,University of South China, 4Shanghai Diabetes Institute, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center for Diabetes,Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

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Transformation Of UAS-cDNA/ORF Plasmids From CRISPR-Based Modular Assembly Into E. Coli:1. Pre-chill 10 μL Tips And 1.5 ML Tubes In An Aluminum Cooling Block On Ice.2. Thaw Competent E. Coli Cells On Ice For 5 Minutes.3. Add 10 μL Of Competent Cells To Each Pre-chilled 1.5 ML Tube.4. Add 1 μL Of The UAS CDNA/ORF Plasmid Products To Each Tube.5. Gently Swirl The Tubes To Mix And Incubate On Ice For 30 Minutes.6. Heat Shock The Cells At 42°C For 1 Minute.7. Immediately Chill The Tubes On Ice For 2 Minutes.8. Add 100 μL Of Pre-warmed (37°C) SOC Medium To Each Tube.9. Incubate The Tubes In A Shaking Incubator At 250 RPM And 37°C For 1 Hour.10. Warm The LB Plates Containing The Appropriate Antibiotic To 37°C.11. Spread The Cells Onto The Warm Plates And Incubate At 37°C Overnight

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