I am trying to isolate many plasmids for a project (ranging from 6.6Kb to 6.2Kb), and the plasmids have a pUC1 ori (high copy number from what I can tell), and Amp resistance. I am using transformed Dh5a cells and am currently growing the bacteria at 37C at 225RPM in 125mL in a 200mL flask with 125 uL of Amp (100ug/mL working conc.) for 16-18 hours (should be shorter from what I read), these conditions were told to me by a PhD student in the lab. Those liquid cultures were inoculated directly from a glycerol stock (I know, bad practice and I am currently making new streaks to make new liquid cultures from those to try for higher yields). I have also recently learned that the protein product of AmpR, B Lactamase, is secreted, possibly spelling trouble if the glycerol stock or liquid culture is contaminated with satellite colonies, another reason I am re-streaking my cultures before liquid culture. When I do a midi prep (Promega Pure Yield) however, I am only getting at best about 130 ug/mL, and the worst have been around 10-30 ug/mL. My gels look pretty good regardless of the outcome however, good plasmid isolation majority in SC conformation and maybe 10% in open/nicked, no gDNA, 260/280 and 260/230 rations are good. From what I have read, high copy number plasmids at this much volume of culture should be giving me much much higher yields. Does anyone have experience with plasmids like this and can help me? Can my culture conditions be changed? Please, any help you can offer would be greatly appreciated! Thanks!
There are a few different issues. First of all you have way too much medium in your flask for good aeration. A rule of thumb is to use only 25% of the flask volume. So for a 200ml flask you should use no more than 50 ml of culture, otherwise the cells are not well aerated and won't grow to the expected density.
Secondly, don't worry too much about the beta-lactamase issue, generally these plasmids are very stable. However it is always safest to pick from a single colony. Third, pay close attention to your midi prep protocol. One common issue I have frequently observed is that the cells are not lysing well. When you add the lysis buffer be sure the solution really goes quite clear otherwise you still have a lot of unlysed cells.
Finally, saying that you have a concentration of 130ug/ml is not useful, that is a concentration but without knowing final volume doesn't determine whether you had good yield or not. If you have 100ul at that contraction it would be terrible, however 1ml at that concentration is a bit low whereas 10 ml at that concentration would be excellent.
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Michael, thank you for your advice. My concentration from above was 130ug/mL in 500uL. I have successfully isolated single clones for my bacteria which I will use going forward. I just isolated a new batch today and got a concentration of about 370ug/mL (average of 3 samples) but the gel didn't look so great, I saw a low of genomic DNA. Other than being more gentle in the lysing process, do you know of any other causes of gDNA breaking? Could it have something to do with culture conditions? The 3 liquid cultures appeared quite foamy when I stopped them, and they were grown in the flasks with the baffling at the bottom to improve aeration. I read this is normally due to lysed cells releasing proteins that can stabilize the bubbles.
Unless you really need more DNA, I wouldn't worry so much about your yields, There are many explanations. However having a lot of contaminating gDNA is more of an issue. Obviously being more gentle upon lysis is critical but it could also be something in cell growth. If you have a lot of cell lysis going on during the growth that could contribute.
When you add the lysis and then the precipitating buffer, I rotate and invert the tubes gently. Don't shake or vortex.
I would suggest trying normal (not-baffled) flasks using 25% of the defined volume, and grow only overnight. Pick a fresh colony from the streak plate in the morning, grow a 5 ml starter culture during the day and then dilute that into the flask at the end of the day and harvest first thing in the morning.
The plasmid size is ok and DH5a is a standard strain that works well.
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