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Alanine scanning by site-directed mutagenesis

KH Katarzyna Z. Haza
HM Heather L. Martin
AR Ajinkya Rao
AT Amy L. Turner
SS Sophie E. Saunders
BP Britta Petersen
CT Christian Tiede
KT Kevin Tipping
AT Anna A. Tang
MA Modupe Ajayi
TT Thomas Taylor
MH Maia Harvey
KF Keri M. Fishwick
TA Thomas L. Adams
TG Thembaninkosi G. Gaule
CT Chi H. Trinh
MJ Matthew Johnson
AB Alexander L. Breeze
TE Thomas A. Edwards
MM Michael J. McPherson
DT Darren C. Tomlinson
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To assess the importance of each residue in the Affimer variable regions point mutations to encode sequential alanine residues were introduced by QuikchangeTM site-directed mutagenesis. Reactions contained 1× KOD polymerase reaction buffer, 0.2 mM dNTP, 2 mM MgSO4, 0.3 μM of forward and reverse primer, 10 ng DNA template, and 1 U KOD polymerase. PCR amplification consisted of 30 cycles of 20 s at 98 °C, 10 s at 68 °C, and 3.5 mins at 70 °C. Samples were digested with Dpn I for 1 h at 37 °C and introduced by transformation into XL1-Blue super-competent cells. DNA was extracted using QIAprep Spin Miniprep Kit as per the manufacturer’s instructions and mutagenesis was confirmed by DNA sequence analysis (Genewiz).

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