Hello all, I'm attempting to get a stable strain of E.coli carrying a plasmid I'm working with. I've performed transformations using the Peg/MgSO4 method, selected on Ampicillin, and I have miniprepped ten candidate colonies for the plasmid.
The attached picture is an enhanced capture of the ensuing gel for candidates 2-10. Because I haven't yet ordered any restriction enzymes, I ran the minipreps as-is, expecting to see the typical three-banded pattern of relaxed/partially relaxed/supercoiled plasmid. Instead, I'm seeing only one band in each lane, which in most of the candidates corresponds to the expected 6160 base pairs. Conditions: - The strain is derived from a chemically competent Top10 kit, and is presumed DH10B. - Cells were grown in LB prepared in-house (using bromelain-treated soy protein rather than casein hydrolysate/tryptone) - Minipreps were performed using a spin-column kit, and extracted in the provided T.E. - The gel is 1%, in-gel staining with "SafeView" from nbsbio.co.uk (with added safeview to the buffer to enhance staining) - Gel was run at 100V to clear the wells followed by 85V. - Illumination was by blue LEDs, filtered by orange perspex (it's a Pearlbiotech.com rig designed for sybr-safe) NB: Safeview is designed for UV transillumination, but does excite at blue and emits green. Sensitivity is consequently less than ideal, but bands are quite crisp to the naked eye; the picture was taken with a camera that doesn't have granular enough control over exposure or focus to optimise. Even to the naked eye, there don't seem to be extra bands. Does anyone have suggestions as to why I'm seeing apparently relaxed DNA? I am pretty confident that the DNA comes from the plasmid I used to transform: I ran negative transformations with the deionised water I've been using, and got no colonies at all. The transformed plates had plenty. -- letters.cunningprojects.com twitter.com/onetruecathal http://www.indiebiotech.com
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