Abstract
An improved method to identify the T-DNA insertion site in transgenic Arabidopsis thaliana (Columbia ecotype) genome was presented. Firstly, the pre-adaptor was amplified by PCR from the plasmid pLASC11.12.8 and digested by HindIII to produce the adaptor. After treated with calf intestine alkaline phosphatase, the adaptor was ligated to the genomic restriction digested fragment with the same restriction endonucleases. Then two rounds of PCR (nested-PCR) were carried out and an unknown sequence between the T-DNA and the adaptor was amplified. Further analysis would reveal the accurate site of T-DNA insertion into transgenic A. thaliana genome.
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Abbreviations
- CIAP:
-
calf intestine alkaline phospbatase
- EtBr:
-
ethidium bromide
- LB:
-
left border
- PCR:
-
polymerase chain reaction
- pre-adaptor:
-
PCR product to produce the adaptor
- RB:
-
right border
- T-DNA:
-
transferred-DNA
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Huang, S.C., Liu, H.Z., He, G.H. et al. An improved method to identify the T-DNA insertion site in transgenic Arabidopsis thaliana genome. Russ J Plant Physiol 54, 822–826 (2007). https://doi.org/10.1134/S1021443707060155
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DOI: https://doi.org/10.1134/S1021443707060155