GFP 96-well black plates; plastic lid, sealer or paraffin oil to cover? - (Mar/09/2012 )
Dear Scientists
I am expressing a GFP protein (GFP mut-1 gene) in a Gram positive host strain.
For this I am using an Appliskan plate reader, which is a top reader for GFP, bottom reader for photometric OD600nm readings.
I have 96 black well plates, with clear bottom for this purpose, however no sealing foils or caps come with these. The typical reagents in each well would be broth, antibiotics, cells and an inducer.
I am concerned about background or autofluoresence which might arise from using plastic lids, condensation from plastic sealers and the potential contamination (and autofluorescence and potential mess) from using paraffin to cover the wells.
Has anyone done something similar to this? Can you recommend any sealers or plate cover which will not interfere with the flouresence reading? Is there another way to do this that I have not thought of?
I cannot switch to bottom reading with this instrument also.
Any advice or links to techniques would be much appreciated.
Regards
Chris
Most plate readers require that the lid is off for accurate measurement of the OD and/or fluoresence. This is to prevent scattering of the light before it hits the sample.
Thank you for replying Bob1.
Usually I would carry out endpoint readings so not an issue to place a sealer on and read without the lid. However for these GFP assays I would be doing kinetic readings over 24 hours, and incubating within the plate reader. With agitation I am concerned about contaminating my own readings and others in the lab if I was to incubate overnight with no lid or sealer.
Is it possible I am just being overly cautious? If most people do the same type of experiments with no sealer or lid then I am willing to give it a shot!
Regards
Chris
Real-time PCR people use transparent plastic sealers for their plates, you could try those. Check your microplate reader manual, it may have some sort of description of what you can and can't use in the reader.