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Construction of plasmids

QX Qingquan Xiao

Last updated date: Jun 4, 2025 DOI: 10.21769/p2823 Views: 330 Forks: 0

original An abbreviated version of this protocol was published in Science Translational Medicine in Jul, 2022
Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor
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  1. Restriction Enzyme Digestion:

     

  2. Backbone3 μg
    10X FastDigest Buffer5 μL
    Enzyme 12.5 μL
    Enzyme 22.5 μL
    ddH2OUp to 50 μL
  3. Reactions were incubated at 37℃ for 3 h.
  4. Separate the digested DNA fragments using 1% Agarose-gel.
  5. Purification of DNA fragments use kit, e.g. OMEGA or Vazyme.
  6. Measure DNA concentration with a spectrophotometer.
  7. T4 ligation:
  8. Linear Vector30 ng
    10X T4 Buf.1 μL
    DNA fragment60 ng
    T4 DNA ligase1 μL
    ddH2OUp to 10 μL
  9. Keep reaction at 22℃ for 1 h.
  10. Transform 5 μL of ligation reaction into chemical competent E.coli cells such as DH5α or TOP10.
  11. Selection colony at LB agar plate with antibiotic for over night at 37℃.
  12. Picking 2-4 colonies and confirmed by Sanger sequencing.

 

 

 

 

Copyright: © 2025 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Xiao, Q(2025). Construction of plasmids. Bio-protocol Preprint. DOI: 10.21769/p2823.
  2. Xiao, Q., Xu, Z., Xue, Y., Xu, C., Han, L., Liu, Y., Wang, F., Zhang, R., Han, S., Wang, X., Li, G., Li, H., Yang, H. and Shu, Y.(2022). Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Science Translational Medicine 14(654). DOI: 10.1126/scitranslmed.abn0449

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