2 replies to this topic

[lovenergy]

Hello

I've recently received a plasmid and need to grow up large quantities for animal injections. I transformed the plasmid into E.coli DH5-alpha and plate out the bacteria on ampicillin plate. Then I selected several big, isolated colonies from the plate and made a 10ml starter culture. These starter cultures all gave reasonable yield (100ul of ~300 ug/ml per miniprep) with expected insert. We thus made a glycerol stock from the remainder of these starter culture and use them for later scale-ups. But the problem is, ever since that first miniprep, my plasmid yield had dropped to only 50-60 ug/ml.
Qiagen and Promega handbooks says that you should always start from a fresh colony from the plate, make a starter culture, then scale-up. My boss said they've always freeze down some of the first starter culture and keep as glycerol stock for inoculation later, and it never had much problems.
The plasmid (pcDNA3) is ~8kb with a ~3kb insert. I'm a little suspicious that the vector is a low-copy as I ran a sequence analysis, the ori is pBR322. But the original yield was quite good.
I'm really puzzled about what could have happened? How could I increase the yield to that original level?

Thanks everyone!

[truta]

lovenergy, on 20 May 2011 - 04:20 AM, said:

Then I selected several big, isolated colonies from the plate and made a 10ml starter culture. These starter cultures all gave reasonable yield (100ul of ~300 ug/ml per miniprep) with expected insert. We thus made a glycerol stock from the remainder of these starter culture and use them for later scale-ups. But the problem is, ever since that first miniprep, my plasmid yield had dropped to only 50-60 ug/ml.

Just to make sure, are you adding ampicillin to your liquid media as well? They might lose the plasmid if you don't keep the selective pressure.

[lovenergy]

truta, on 26 May 2011 - 12:22 AM, said:

lovenergy, on 20 May 2011 - 04:20 AM, said:

Then I selected several big, isolated colonies from the plate and made a 10ml starter culture. These starter cultures all gave reasonable yield (100ul of ~300 ug/ml per miniprep) with expected insert. We thus made a glycerol stock from the remainder of these starter culture and use them for later scale-ups. But the problem is, ever since that first miniprep, my plasmid yield had dropped to only 50-60 ug/ml.

Just to make sure, are you adding ampicillin to your liquid media as well? They might lose the plasmid if you don't keep the selective pressure.

Yes, I'm adding 2x ampicillin to the medium, final concentration 200 ug/ml. I tried starting from a fresh colony on the plate, made a 10 mL starter culture, then scaled up,the result was much better. It seems like the glycerol stock contained a lot of 'bad' or 'unhappy' bacteria...also the yield was better when the culture is well aerated.
Cheers!