Agilent Technologies
QuikChange II XL Site-Directed Mutagenesis Kit
200521
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Overall research of this project was to study the protein targeting to flagellar membrane. The specific goal was to see if positive charged residues (such as lysines) in our protein of interest are necessary for the flagellar targeting. For this reason we decided to use site directed mutagenesis kit to mutate all lysine residues in our the N-terminus of our protein to alanines. This kit was used because of numerous other successful uses in the past.
Immunofluorescence
Trypanosoma cruzi DNA
Oligo primers were first designed to mutate all three lysines with alanines. All plasmids were purified using Qiagen kits (Qiagen, Gaithersberg, MD), and the relevant regions of all constructs were sequenced to assure accuracy. The construct was used than to transfect the trypanosoma cells and lastly immunofluorescence was used to detect if protein localization was altered as a result of mutagenesis.
Oligos rich in G and C residues that are approximately 35-45 nucleotides in lenght are the most optimal.
When we mutated three positive lysine residues to neutral alanines our protein of interest no longer localized to the flagellar membrane.
None
Ease of use
I successfully used this kit on numerous occasions to introduce site directed mutations, insertions and deletions always with positive outcome. This kit is highly recommended.
Product Name: QuikChange® II XL site-directed mutagenesis kitSupplier Page