Hi,
I am not familiar with the Brimbroim Dolly method and couldn't find anything on the net. How many different bands do you see? Plasmids can run in multiple sizes depending on the conformation. Google nicked, linear, circular single stranded and supercoiled plasmid sizes. Could it be your extraction method is too harsh?
Hi,
I am not familiar with the Brimbroim Dolly method and couldn't find anything on the net. How many different bands do you see? Plasmids can run in multiple sizes depending on the conformation. Google nicked, linear, circular single stranded and supercoiled plasmid sizes. Could it be your extraction method is too harsh?
Dear Reshma,
I am not aware of the method either but Corelia is absolutely right about the the different conformations. Another justification is presence of multiple plasmids, each having different conformations. Try using the mini prep kit by Qiagen, if the results match you can eliminate troubleshooting the Brimbroim Dolly method.
Regards
Thank you all for the answers...So can I actually say it as multiple plasmids of different sizes??My aim is plasmid profiling in Vibrios....I have got bands in size ranging from 3 to 33kb....So can I conclude it as multiple plasmid bearing strains......??
There is a slight correction actually the method is Birnboim and Doly, 1979....sorry it was misspelt.....
I found this article about plasmid isolation in Vibrio: http://www.ektodermaldisplazi.com/journal/Journal2010/Vol3_No1/9M_P_Manivasagan2.pdf
They found up to 3 plasmids in one strain.
Do you use an established Vibrio strain or did you isolate the bacteria from a water or food sample? If the later case is true you could have a mix of Vibrios and other plasmid containing bacteria or more likely a mix of different Vibrio strains. I would suggest you streak out your Vibrio cultures and pick several single colonies and do plasmid preps. Hope this helps.
Thank you Cornelia...But all my isolates are already pure cultures and confirmed by PCR with species specific primers....Then can be it multiple plasmids?
Have you tried profiling them with restriction enzymes? It might give you a better hint as to what's going on.
Have you looked at table 4 in that paper? For Vibrio cholerae the list 5 different strain with 1 to 3 plasmids. All the same species, just different strains. Vibrio parahaemolyticus: 5 strains, 1 and 2 plasmids etc. You will also find the sizes of the plasmids in this table, they range from 35 to 112kb. To answer your question: More than 1 plasmid and/ or mix of multiple strains (each with different plasmid) of the same species.
I've used the Birnboim and Doly prep for years, so I don't think the problem is the plasmid prep. It is pretty hard to diagnose without a picture, and also need to know if you digested your DNA or are running it uncut. Remember that if you isolated plasmid DNA and run uncut on a gel you will get many different sizes representing plasmid monomer, dimer, trimer etc due to recombination, in addition to getting supercoiled and nicked plasmid. But upon digestion they will all linearize to identical size.
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