celvas, on Jun 17 2004, 08:45 AM, said:
Lot of thanks for your kind reply. It helps me for good practical techniques. Let me ask you an other question:
As the plasmid produced by thawed-refreeze glycerol stock has been sequenced, I need to work with it. The question is:
If I streak an agarose plate containing selective antibiotic and the bacteria growth, can I think that these bacteria are suitable for quality plamid extraction? Or do you recommend me for sequencing an other clone stock?
hi
you don't have to use agarose to make a plate, agar is the one people usually use, unless you have special purpose.
bacteria bearing a plasmid grow do not mean they are good for high quality plasmid isolation - of course it depends on what purpose you use it for - sequencing would need a higher quality than general cloning. sometimes it also happens that one bacteria is good for plasmid A but not good for plasmid B, plamsid size, or copy number also play a role. different host (bacteria) have different genotype which would affect you plasmid "quality" and yield. for example some bacteria have wt methylation gene, say dam gene for methylating adenine of GATC, no matter how pure your plasmid DNA is, you can't cut it using restriction enzyme like BclI which recognizes TGATCA, b/c BclI is sensitive to this methylation. Having you plasmid DNA methylated is not always bad, sometimes people need methylated plasmid to transform a special host, other the unmethylated plasmid would be restricted.
the newer E. coli host like DH5alpha, DH10B, XL1-Blue... have many wt genes inactivated which make your plasmid DNA easier to be purified, and usually fit most, if not all, of your work.
hope this helps,
Mike
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