I isolated some unknown resistant bacteria and extracted their plasmids. Can I use a 1 kb ladder to detect the plasmids?
Dear Nayereh Younessi
you can predict this plasmids size , in my research I used 1 Kb for staphylococcal genomic and plasmid DNA
Dear Younessi,
If it is an unknown organism, then it is better to use 1Kb and 100bp ladder on both sides of the isolated plasmids lane as already suggested. Once you have estimated the sizes, you can then choose your marker accordingly. You can also use 100bp plus ladder which has additional marker sizes above 1 kb too. It gives a varied range from 100 to 3000bp. Here is the datasheet.
Hope it helps!
https://tools.thermofisher.com/content/sfs/manuals/MAN0013009_GeneRuler_100bp_Plus_DNALadder_250ug_UG.pdf
I red some papers which used DNA hyperladder or digested λ DNA for plasmid detection. none of them used 1 kb ladder that s why I got uncertain.
There are multiple ladders you can use and it will depend on the size. The 1 kb plus ladder of 100 bp ladder would work for smaller plasmids. Another ladder you could use is the supercoiled DNA ladder which ranges from 2-16 kb. This is the one I have used most extensively with plasmids, especially in capillary electrophoresis and has worked well for sizing. Digested λ DNA works and has a range of 125 bp to 23 kb. So a lot will depend on the size of your plasmid. For an unknown size perhaps the digested λ DNA may be best to start with as it has the larger range.
One thing you may want to keep in mind is the configuration may cause some variation in migration in a gel. However in slab gels this variation is small and any of the above ladders will give you an approximate size.
Plasmids larger than 1kb can be detected by gel electrophoresis if I use 1 kb ladder?
Dear Younessi,
DNA larger than 1 Kb also can be detected by using 1Kb ladder as it has higher molecular weight marker till 10Kb as well. But when you isolate the plasmids from an organism, it tends to remain in supercoiled form as covalently closed circular DNA. So, it'll run faster in an agarose gel than its linearised counterpart. So, exact base pair length cannot be determined until and unless you linearise it with restriction digestion. But definitely you can get an estimate of the size range of the marker you need to use.
Hope it helps!
I have 2 related questions. Can I plated bacteria on solid media plus antibiotics and then use these bacteria for extraction their plasmids? In the liquid media most of my bacteria don t grow up even without the use of antibiotics.
In stage 1, in most cases I have to vortex the samples a long time but the colonies still are visible . what should I do?
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