8 replies to this topic

[benjibin]

How do I know my interest strain(bacteria,gram+) having plasmid?and If it has,how do I get it?
Thanks

[leahf]

You need to do a plasmid isolation from your bacteria and run it on gel. easiest is to use a commercial kit (we use Qiagen or Roche , there are probably others as well) which has complete instructions and takes about an hour. you will need several ml of pure culture of your bacteria .
Good Luck!
Leah

[benjibin]

My strain is micrococcus and I haven't the protocol for the extration of its plasmid.Do you have one?

By the way, can you tell me which kit is better about it?
Thanks!

[leahf]

sorry, i didn't notive you said gram plus!
All protocols i know have been designed for coli which is gram -. i don't think it should make much difference expect you'd probably have to add  a step to break down cell wall - lysosyme might do it. also, if your cells clump that might also be a problem.
i would try searching through Google or Web of Science for articles that have isolated plasmids from micrococcus, and look at their methods.

About the companies  - I use the "Qiaprep spin miniprep kit", from Qiagen for isolating plasmids from vibrios (which are gram-negative) and it works great (though it was designed for E.coli). Roche is also very good but i have never tried it for anything other than coli. other companies of good reputation which probably have kits are Sigma and Promega. All these companies have technical assitance departments about their products and you can ask them about gram plus. usually its best to find their representatives in your own country and work through them.

[Charon]

If you got many strains to test i'd recommend Eckhardt lysis in contrast to a fully blown plasmid extraction.
Especially cause it's cheaper

[yzhou715]

It could a very difficult job.  If you think that an interesting gene may be encode by the plasmid, it may be worth time to try different methods.  In your case, you do not know the size of a plasmid, some bacteria have real big plasmid (>100kb).  Most of the methods are not good for that.

One sugestion is using tranditional method and then try to further isolate plasmid from a crude DNA preparation.

[desty]

do you get the plasmid??

[hanming86]

Try using the traditional method. The column has a limit. Big plasmid can bind to column but might not elute out. If u look at even the gDNA extracted using column, u will notice that their overall size is approx 20kb. that seems to be the limit of column.

It would not harm to try out the commercial one first.
But then again traditional method seems to be safer in this situation.

[molgen]

Every plasmid has an origin of replication.
You can try to design primers to detect that part of the plasmid.

I've never tried it but it might work.