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Construction of plasmids
Last updated date: Jun 4, 2025 Views: 350 Forks: 0
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Restriction Enzyme Digestion:
Backbone 3 μg 10X FastDigest Buffer 5 μL Enzyme 1 2.5 μL Enzyme 2 2.5 μL ddH2O Up to 50 μL - Reactions were incubated at 37℃ for 3 h.
- Separate the digested DNA fragments using 1% Agarose-gel.
- Purification of DNA fragments use kit, e.g. OMEGA or Vazyme.
- Measure DNA concentration with a spectrophotometer.
- T4 ligation:
Linear Vector 30 ng 10X T4 Buf. 1 μL DNA fragment 60 ng T4 DNA ligase 1 μL ddH2O Up to 10 μL - Keep reaction at 22℃ for 1 h.
- Transform 5 μL of ligation reaction into chemical competent E.coli cells such as DH5α or TOP10.
- Selection colony at LB agar plate with antibiotic for over night at 37℃.
- 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:
- Xiao, Q(2025). Construction of plasmids. Bio-protocol Preprint. bio-protocol.org/prep2823.
- 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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