4 replies to this topic

[bry512]

The case is that the SV40 plasmid which I used for luciferase assay is nearly used up, and I need to midiprep more.
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?

[bob1]

If you grew them from a glycerol stock, all the colonies should be the same. However, if you are transforming and selecting on a plate fresh each time - 1) you should do it from glycerol - much faster. 2) it doesn't really matter how many copies of the plasmid are in each bacterium, you will still have billions of copies at the end.

[bry512]

thank you very much for ur suggestion, but i still donot understand how to do~
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?

[bob1]

Bacteria can be kept at -80˚C in "glycerol stocks" which are ordinary liquid cultures of bacteria transformed with your plasmid and then with glycerol added to about 15% volume per volume and snap frozen.

Protocol:
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.

[bry512]

Thank you very much for your protocol.