I transformed a plasmid which self-expressed mCheery fluorence in cells, but it seems to express weakly in E.coli. Therefore, the FACS could not seperate the cells with and without plasmids well. These pictures are cells with plasmids and the blank control (cells without plasmids). I wonder the reason of this problem or if there are methods that could enhance the mCheery fluorescence?
We will need more details on your protocol. How long after transfection did you wait before trying to sort the cells? What brand of apparatus and settings did you use for the sorting?
Robert Adolf Brinzer Thank you for answering my question.
This is experimental evolution and 10 μL culture was transfered to 980 μL LB medium with 10 μL low-dose antibiotics every 24 hours. The ratio of cells carrying the plasmids was identified every 6 days. Fluorescence was monitored by the Beckman CytoFLEX SRT. Overnight cultures were diluted 1:100 with PBS in 5 ml culture tubes for loading on the flow cytometer. The channel of emission detector wes set as P1: 561-Yellow (Y610/20 nm) for PE-mCheery. For each sample, 30,000 cells were collected. And the gain for mCheery was 1000.
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