Hello everyone,
I need genomic E. coli DNA as a template for later PCR. I have a protocol how to isolate genomic DNA and it says I have to prepare an overnight culture of XL1 blue cells. We don't have any plates or glycerol stocks of untransformed XL1 blue cells. Since the genomic DNA is still present in transformed cells, I could use any glycerol stock of transformed XL1 blue cells, is this correct? Also, I don't have to use the antibiotic resistance of the introduced plasmid, do I?
Moreover, I am wondering whether I could just put bought competent XL1 blue cells (which you normally use for transformation) into LB medium? I can't find any information on this last question.
Thanks!
Below link can help you.
https://www.chem-agilent.com/pdf/strata/200249.pdf
You can use your transformed cells, however, part of the population will retain the plasmid even without the antibiotic selection and you will get the plasmid sequence from your sequencing. You can do the following:
1-passage the cells for two or three overnights then plate and screen for plasmid loss (streak single colonies on both LB and LB + antibiotic and the ones that have lost the plasmid will only grow on LB hence are your plasmid free parent strain). Keep in mind this depends on the plasmid, some plasmids are very very stable and will not be lost this way. Also you might get some mutations from repeated culturing.
2- Do as you thought and buy the competent cells. Just use a tip to scratch the top of the frozen competent cells and dump the tip in LB, the cells will grow fine and you still get to keep your cells competent if you re-freeze quickly.
P.S XL1-blue contain an F episome and that will not be lost easily even if you don't select for the plasmid borne tetracycline resistance and you will get F sequences.
- Badges
- Science topic