How to evaluate SV40 expression level
Posted 04 October 2009 - 07:29 PM
in this case, shall I pick several single colonies to select one which has the highest expression level, or just scrape several? If I need to pick the single colony, is there any easy way to evaluate which clone has the highest expression level?
Posted 05 October 2009 - 03:34 PM
Posted 05 October 2009 - 07:52 PM
what do u mean by "grow them from a glycerol stock"? Now I only have the remaining several ul of SV40 plasmid, what shall I do next? Could explain more in details?
Posted 07 October 2009 - 03:26 PM
1) take plasmid and transform it into bacteria (heatshock, electroporation, etc. any old method will do)
2) spread on plate with selective antibiotic and incubate appropriately
3) pick colonies and test for presence of plasmid and correct sequence
4) grow liquid colonies
5) take 850 ul of liquid culture and add sterile glycerol to 1.0 ml
6) snap freeze (dry ice/methanol or liquid nitrogen)
7) store in -80˚C freezer
These stocks will keep for 20+ years and when you need some more take the stock out of the freezer and scratch the top with a loop (making sure that you keep the stock frozen), then streak out on a plate and pick and grow. Voila, an indefinite supply of plasmid.