4 replies to this topic

[ranvi]

I am doing plasmid prep regularly in maxi and giga scale, but I  never  calculated cell density, I grow cells at least 15 hrs. so far no problem. All of sudden I am experiencing such  a low yield

previous yield on Maxi using invitrogen kit is around 1mg now not even 300ug
Giga (invitrogen) gave around 10mg but now not even 1mg

what could be the reason?.

I used just home source vacuum, but for the last Giga prep I used vacuum controller, is that could be the reason?

also how to calculate the density on this grown cultures and how much it should be?

Any help is appreciated/

Thanks

[protolder]

Hola, I never have used vacuum devices for maxipreps, but I think there is not difference between an oil pump or a water trop for instance. I suppose that you have seed your cultures from glicerol -80 stock, if non this could be a reason for the lost of yield. As everybody do you seed overnight cultures in LB. The optical density that it reaches is about 3ud.Take 100ul of culture and 900ul of PBS or saline solution (NaCl 9g/l) and read the absorbance at 6oonm with water as reference. The spectrofotometric value is multiplied by 10 and this is the OD of the culture. Good Luck

[HomeBrew]

Does the culture that gave you a low yield contain the same plasmid as the culture that gave you a high yield?  If not, perhaps the high-yield culture contains a high copy number plasmid and the low-yield culture contains a low copy number plasmid?  Or requires a different antibiotic for selection (which might effect the end-point density of the culture)?

Were both the high-yield culture and the low-yield culture grown in the same media and under the same conditions?

Were the A260/A280 ratios pretty much the same for both cultures?  Remember, the spectrophotometer measures anything that absorbs the wavelength used -- a dirty sample will read higher than a clean sample, without necessarily containing more plasmid DNA.  Do equivalent amounts of the samples appear different on an EtBr-stained gel?

It would be useful to have culture ODs to compare, to see if it's a culture growth issue or a plasmid extraction issue...

[ranvi]

I used the same glycerol stocks to grow them, and used Vacuum only for GIGA not for maxi  

What the OD600 at the time of harvesting?


Thanks

[protolder]

Hola, about 3 OD units (in LB). Have a good day