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Our lab investigates how the diversity of innate immune system genes impacts immune system function. Specifically, we study how polymorphisms of NK cell receptors called killer-cell immunoglobulin-like receptors (KIR) alter their NK cell function. We use an insect cell-expression system to produce recombinant proteins for use in a cell-free binding assay. To produce these proteins, we use a baculovirus transfection system in which our target gene is combined with linearized baculovirus and transfected into Sf9 insect cells. For this protocol to be successful, exactly 2ug of target gene DNA (in an appropriate display vector) must be added to 0.2ug linearized baculovirus. Given that we are often transfecting up to 20 samples at a time, establishing the concentration of target gene DNA in a timely fashion is of importance to us. The NanoDrop 8000 provides this utility for us – we use it primarily for the determination of nucleic acid concentrations – the fact that is has 8 channels that allow simultaneous measurement of 8 samples facilitates this process without compromising the accuracy of the measurement made.
Determining nucleic acid (DNA) concentration for recombinant protein expression.
Gene of interest cloned into an appropriate display vector (e.g pacGP67a)
Libraries of genes, each in an insect cell display vector, are cloned and individual colonies isolated from E.coli. Each colony is expanded and the target DNA is isolated via mini-prep and sequenced. The NanoDrop is then used to determine the concentration of each of the samples. The NanoDrop is controlled remotely using software run on an attached PC. When first booting up the NanoDrop for the day, the spectrophotometer should be cleaned with distilled water and dried using lab tissue. The instrument is then blanked using PCR grade water. When determining the concentration of multiple samples, a multi-channel pipette can be used to transfer DNA samples from an 8-well strip onto the NanoDrop. One ul of each sample should be transferred onto each pedestal. The identity of each sample can be recorded and stored using the software provided. The NanoDrop determines the concentration of each DNA sample quickly (around 10s) and also provides the A260/280 ratio, which is an indication of the purity of the sample.
The NanoDrop 8000 should be calibrated every 6 months (or more often depending on frequency of use) to ensure that the measurements made are accurate.
The NanoDrop provides a quick and accurate determination of the concentration of nucleic acids. We determine the accuracy of the instrument with samples of DNA at known concentrations. The variation between measurements of the same sample (repeated) is also minimal. In addition, the calibration procedure also lets us know that the instrument is operating within certain accuracy parameters.
User-friendly software that allows the user to run the NanoDrop and to record and save the concentrations of multiple samples (up to 96). A260/280 ratio allows simultaneous determination of the purity of each sample. Instrument boot-up time is minimal unless blanking is required for the first user of the day. The 8 channels allow the user to determine the concentration of multiple samples quickly.
None.
Product Name: NanoDrop 8000 SpectrophotometerSupplier Page