Jas Sanghera, Joby Jenkins, Rob Lewis, Chloe Milburn
A prerequisite for efficient high throughput protein crystallisation screening is the accurate pipettingand positioning of the low volume drops used in hanging and sitting drop setups. Solutions exist for low volume pipetting, however, the variable viscosities of protein and reservoir/screen solutions present significant challenges for many liquid handling systems. The mosquito® (TTP LabTech) offers fast positive displacement pipettingfor accurate and reproducible aspiration and dispensing throughout the 50 nL-1.2 µL range, producing CVs of <8% at 50 nLirrespective of viscosity. Mosquito’s micropipettes are also disposable, thus guaranteeing zero cross-contamination where required.
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We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.