A retroviral sgRNA library was designed targeting all the sites bearing PAMs throughout a 20-kb region surrounding the Foxp3 locus (from 12.5 kb upstream to 7.5 kb downstream of the transcription start site). Specifically, we first identified all PAM sequences (NGG) within the X chromosome: 7143000–7165000 (mm9). To ensure the target specificity, we filtered the targets with low complexity sequences, such as repetitive ACG and TTTT. We then selected the targets ending with PAM and bearing unique sequence specificity in the mouse genome. In addition, 100 nontargeting negative controls were added to the library (Table S1 ). A library containing 75-nt single-stranded DNA oligonucleotides was synthesized by using a high-throughput method (GenScript) and amplified by PCR with primers binding to the flanking arms according to standard protocols. PCR product was then assembled with BbsI-linearized pSIR-BbsI-Thy1.1 vector backbone (modified from pSIR-hCD2, Cat51143; Addgene) by using Gibson Assembly Master Mix (New England Biolabs). The resulting product was electroporated into Escherichia coli (New England Biolabs) and selected by ampicillin on 16 25-cm2 dishes. Transformation efficiency was quantified to ensure sufficient coverage. Plasmid was extracted by using the CompactPrep Plasmid Maxi Kit (QIAGEN), and the sgRNA coverage was validated by performing high-throughput sequencing.
Compactprep plasmid maxi kit
Manufactured by Qiagen
Sourced in Germany
The CompactPrep Plasmid Maxi Kit is a laboratory equipment used for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a modified alkaline lysis method and silica-based membrane technology to efficiently isolate plasmid DNA.
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Lab products found in correlation
3 protocols using compactprep plasmid maxi kit
1
CRISPR-Based Screening of Foxp3 Locus
2
Plasmid Transfection of SOD1 Variants
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pEGFP-N1 expression vectors containing SOD1WT and SOD1G93A cDNAs were kindly provided by Bradley Turner (University of Melbourne, Melbourne, Australia) [33 (link)]. pFUW expression vectors containing SOD1G85R and SOD1G127X cDNAs were kindly provided by Edward Pokrishevsky (University of British Columbia, Vancouver, Canada) [9 (link)]. Chemically competent DH5α Escherichia coli, kindly provided by Jason McArthur (University of Wollongong, Wollongong, Australia), were transformed with SOD1-containing plasmids using heat shock (42 °C) and colonies resistant to kanamycin (Ameresco, Framingham, MA, USA) selected. Plasmid DNA was purified using the CompactPrep Plasmid Maxi Kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions.
3
Plasmid DNA Isolation and Transformation
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Bacteria of Competent E. coli DH5α strain (NEB® 5-alpha Competent E. coli (High Efficiency) NEB C2987H) were cultured in a liquid LB medium at 37 o C. The bacterial strain was stored at -80 o C as stock in LB medium with added glycerol. Bacteria were transformed with plasmid DNAs using the heat shock method according to the manufacturer's instructions. Bacteria were inoculated on LB agar plates with ampicillin and incubated at 37 o C overnight. Plasmid isolation was performed from selected bacterial colonies with the CompactPrep Plasmid Maxi Kit (#12863, QIAGEN) . The following quality control steps for DNA and virus production have been evaluated by considering the specified acceptance criteria. DNA concentration was measured in ng/µl with Microplate ELISA Reader (FLUOstar Omega) and it was evaluated whether the purity value was between 1.8
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