6 replies to this topic

[tyrael]

Hi all.

i m now working onto some new isolated bacteria on my own and i have no idea that wat kind of plasmid in inside them.
i have extracted out the plasmid DNA and did a gel run.

and it show me with some multiple bands, yes, i know it's common for plasmid DNA due to its conformation (supercoiled, nicked and linear), so it gives me different migration pattern.

my question is,

how do i determine which is the EXACT size of the plasmid that i extracted ? since it migrates differently though at same size ?


pls correct me if i am wrong.
thank you.

[HomeBrew]

The only way to know the EXACT size is to sequence it.  You can get an approximate size by cutting it with a restriction enzyme and seeing what size fragments are produced.  It might take testing with a few different enzymes to find one that cuts into only a few fragments, so you can add the fragments sizes up to get the total.

[Curtis]

yes as HomeBrew said you need to cut your plasmid to give you a linear plasmid then run on the gel again. but my question is that if your bacteria strain is already known, then can't you find that plasmid sequence at Genebank or somewhere to figure out what RE would give you only 1 linear band?

[T C]

Hey,

Run standard vectors without digesting and compare their mobility and estimate aproximate size of yr plasmid.

Hope it helps.  

Best,
TC

[tyrael]

Quote

but my question is that if your bacteria strain is already known, then can't you find that plasmid sequence at Genebank or somewhere to figure out what RE would give you only 1 linear band?

but different bacteria may possess different plasmid one, right ? then how could i be so sure that which plasmid that it have ?  

[perneseblue]

I agree with TC. Cut the plasmid with a restriction enzyme. And run the DNA fragments with a DNA ladder.

You might get several DNA fragments from the digest. Just sum the sizes of the DNA fragments.

Repeat this for several different enzymes just in case an enzyme does not cut or cuts too frequently.

[tyrael]

   Ok. Thank you all so much! i will proceed with the restriction then. =)