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URL: https://dx.doi.org/DOI:10.17504/protocols.io.2pcgdiw

Authors: Jacob Beal, Cheryl Telmer, Richard Tennant, Paul Rutten

Group: iGEM Measurement

Summary: This protocol can be applied to any strain of cell that can be safely run through a flow cytometer. For clarity, we have written it assuming E. coli DH5-alpha; to apply the protocol to another cell type, substitute the other cell type for any place where the protocol says [E. coli DH5-alpha].This protocol has been written for measurement of GFP, YFP, or other yellow/green fluorescent proteins into MEFL units. To apply it to fluorescent proteins of other colors:Replace BBa_J364001 with a construct for strong expression of the other protein. For blue proteins (e.g., mTagBFP), measure with 405nm excitation and 450nm/50nm emission filter. Units will be MEC30.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.For blue proteins (e.g., mTagBFP), measure with 405nm excitation and 450nm/50nm emission filter. Units will be MEC30.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.For red/orange proteins (e.g., mCherry), measure with 561nm excitation and 610nm/20nm or 620nm/15nm emission filter.  Units will be MEPTR.For far-red / near-infrared proteins (e.g., IRFP), measure with 635nm excitation and 780nm/60nm or 750nm long-pass (LP) emission filter. Units will be MEAPCY7.To apply the protocol to multiple colors, add a positive process control for each color and use one of the tools on the iGEM Measurement Resources page to compensate measurements for spectral overlap.This protocol can be combined with bead-based cell size calibration.

Affiliations: iGEM Measurement Committee, iGEM Measurement Committee, iGEM Measurement Committee, iGEM Measurement Committee

External URL: https://2019.igem.org/Measurement

Version: 1

Publication Date: 2019

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