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Our lab investigates how the diversity of innate immune system genes impacts immune system function. Specifically, we study how polymorphism of NK cell receptors called killer-cell immunoglobulin-like receptors (KIR) alters their NK cell function. We have recently designed a cell-free assay in which we use recombinant soluble KIR proteins bound against a panel of 96 HLA class I antigens. We use site-directed mutagenesis to generate libraries of recombinant KIR proteins that represent the variety found in the human immune system. The gene-of-interest is cloned into an appropriate vector (in our case a baculovirus display vector for use in an insect cell expression system) and we use the Veriti thermal cycler to introduce one or more mutations into the gene of interest using site-specific primers.
Site-directed mutagenesis
Gene-of-interest cloned into an appropriate display vector (e.g., pacGP67a), dNTP mix, TAQ polymerase, forward and reverse primers with desired mutation, PCR buffer, water.
Dilute the target DNA to 50ng/ul with PCR grade water and add 1ul to the following mix: 5ul 10x PCR buffer, 1ul each primer (10uM), 1ul dNTP mix (4uM), 1ul Turbo Polymerase (Agilent, cat #600252). We use the following protocol for our site-directed mutagenesis – when we switched to using the Veriti thermal cycler, we had to extend our extension times considerably (above the recommended 1 min per kb of vector) as the rapid ramping that is a feature of this instrument was reducing the efficiency of our mutagenesis. Our current protocol (with an appx 9kb plasmid and 1kb insert) is as follows: 95°C for 30s, 18x (95°C for 30s, 55°C for 60s, 68°C for 15min).
Increasing the extension period over slightly over the recommended times will increase the efficiency of the site-directed mutagenesis.
The Veriti thermal cycler provides a readily programmable and user-friendly interface for all PCR reactions. The touch screen display is intuitive and allows the user to see the progress of the reaction easily.
The thermal cycler can be easily programmed using the intuitive touch screen. Unlike many older thermal cyclers this process is intuitive and easy to follow, although the boot-up time for the instrument is considerably longer than traditional machines. Previously used PCR protocols are easily stored (ours are stored in chronological order so that recently used protocols are readily accessible) and they can be accessed from the main start-up menu.
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