Site specific mutagenesis can be used to generate mutants in specific residues. The method used here was originally developed by Kunkel (see ref. 1).
Method Article
Generation of deletion and insertion mutants
https://doi.org/10.1038/nprot.2007.85
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mutagenesis
insertion
deletion
Site specific mutagenesis can be used to generate mutants in specific residues. The method used here was originally developed by Kunkel (see ref. 1).
mutagenesis.
For the C-terminal truncation mutants, use single strand DNA of pDRf1-AtAMT1;1 as a template to insert a short DNA fragment coding for a stop codon and carrying XhoI restriction site (5’tgagtcgac).
Remove the remaining part of the Cterminus by XhoI digestion.
For all insertion mutants, use single stranded DNA from pDRf1-AtAMT1;1 carrying a 3x cmyc-tag at the very end of the AtAMT1;1 C-terminus as template.
Insert the C-terminal insertion of 5’atcgatggacgt, encoding IDGR, at positions 443, 444, 445, 446, 448, 455 or 456 into the AMT1;1 protein. For the L3-4 (Ins150) and L5-6 (Ins 231) insertions, insert a 5’ggttggagctcccgggggcgcc DNA fragment, encoding GGAPGGA, at position 150 or 231 into the AMT1;1 protein.
Insertions:
Ins150: …IAER_GGAPGGA_TQFV…
Ins231: …FDN_GGGAPGGA_GRAI…
Ins443: …ILKK_IDGR_LLRI…
Ins444: …LKKM_IDGR_KLLR…
Ins445: …KKMK_IDGR_LLRIS…
Ins446: …KMKL_IDGR_LRIS…
Ins448: …KLLR_IDGR_SSED…
Ins455: …EDEM_IDGR_AGMD…
Ins456: …DEMA_IDGR_GMD…
(1) Kunkel, T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488-492 (1985).
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