Hey there,
I've tried to perform the FPG-Comet with frosted-end slides, but after the 30min-incubation step at 37°C the gels got already very fragile (only once they survived without bigger damage), mostly peeling off finally.
The protocol I've found was suggesting a moist box for the incubation, but it doesn't say, whether it is supposed to be tightly closed or not. (once fully closed box, once slightly opened)
So I wonder whether my slides suffered from too much dryness, too much moist, or just the high temperature.
(A former colleague has worked with fully frosted slides, they seem to be less prone to peeling off from the incubation step.)
Thanks for your help!
Dear Ivan,
these tips could help you,
first coat the slides with normal agarose at 1 %. store at 37º C until use.
- to make gels with the cell suspension use the LMP agarose at concentration of 1 %. The mix of the cell suspension:agarose 2 parts cells:8 parts agarose. eg. 20 microliters of cell suspension mixed with 80 microliters of LMP agarose, or more agarose , 10 mricoL of cells:90 microL of agarose.
- make small gels, eg. a drop of 80 microliters covered with coverslips of 18x18 mm or 20x20 mm, make two per slide. set the gels at least five minutes at 4ºC to make firm gels.
- the lisis step at 4ºC or less to preserve the gels.
- wash the gels with PBS or enzime buffer at 4ºC .
-drop the enzime over the gels and then cover it with flexible coverslips of parafilm.
-incubate in a moist chamber. tightly closed or not is not critical for a 30 minutes incubation.
-after the enzime incubation period wash the gels with electrophoresis buffer at 2-4ºC .
-perform electrophoresis at 4ºC.
Regards, Jorge
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