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تحويل واسع النطاق لبلازميدات UAS-cDNA / ORF من التجميع المعياري القائم على كريسبر إلى الإشريكية القولونية

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01:46 min

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May 17th, 2024

DOI :

10.3791/201692-v

May 17th, 2024

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15 Views

Si Xu^1,2*, Mengyu Chen^1,2*, Guang Chen^1,3, Si Luo⁴, Wen Xue^1,2, Xuelian Liu^1,2, Jiwu Wang^1,2,3, Ping Wei⁴

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

* These authors contributed equally

Transcript

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Transformation Of UAS cDNA ORF Plasmids From CRISPR Based Modular Assembly Into E Coli 1 Pre chill 10 L 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 L Of Competent Cells To Each Pre chilled 1 5 ML Tube 4 Add 1 L 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 L 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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A High-Throughput UAS-cDNA/ORF Plasmid Library Construction Using CRISPRmass

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