plasmid miniprep question - (May/25/2006 )
I am currently undergoing a project to determine which plasmid miniprep kit will be used in a clinical laboratory. I am familiar with the procedure (most of the kits are very similar). However, I was wondering if anyone has tried to use bacteria directly from a plate culture, suspending a number of colonies from a plate in the resuspension buffer. This would be especially helpful in a clinical setting where we could forgo the overnight growth in a liquid medium, in order to do same day analysis.
Any help would be greatly appreciated.
DB
Sometime I grow bacterial cultures for only 6-8 hrs and do miniprep. Saves some time.
I don't see why the heck not, seeing as you're just going to be pelleting the cells down again anyhow. So long as the plasmid's in high enough copy number.
Unless of course, someone who's done this wants to say different.
I have on occasion done minipreps directlt from a culture plate by resuspending growth in the resuspension solution, and proceeding from there. It worked fine.
The only thing to work out is how much bacteria you'll take from the plate, and whether the plasmid's copy number is high enough.
What will you use the plasmids for? If it's just a present or absent question, or you'll use the plasmid for some sensitive application (PCR, Southern, etc.), you'll be fine. If you're trying to clone from the plasmid, you might have to tweak it a bit to recover enough plasmid DNA.
In general though, it should work fine.
Straight from a plate, no problem. Use multiple platings, streaks, etc. if you need more plasmid. Isolating from plates is used often for library amplification., as opposed to growing a culture in liquid, so a colony or colonies do not take over the culture and become over-represented in the final isolation.
To find out the concentration of cells needed from a plate: scrape some, resuspend in M9 or saline, OD and determine the cell number. Is it too much or not enough as per the kit instructions? - scrape more or split the sample...