3 replies to this topic

[Smog187]

HI,

I'm currently trying to isolate plasmid DNA from DH5-alpha cells using the QIAgen Quick-Lyse Kit, and I have been consistently getting low DNA concentrations (20ng/uL) while another member of our lab is using XL-10 cells and is having the same problem.

We've tried multiple variations to only get the same results, such as:
We thought it might be the kit, so we used another lab's kit (columns and reagents) to only get the same result.
We then tried trying Biorad's Quantum mini-prep kit to once again get the same problem.
Next we thought it might be our LB so we made new LB and even borrowed another lab's LB to once again get the same result.
We messed with the growing times. We tried 12 hours, 16 hours, and 18 hours and even measured the ODs but still the same problem.
We tried using different amounts of LB (3mL, 5mL, 10mLs.) and that didn't work.
I even increased the concentration of ampicillin, just in case the large amount of bacteria were leading to the degradation of ampicillin and we were growing bacteria that didn't have the plasmid.
Lastly, I did a fresh transformation in a desperate hope that it would fix the problem. But it didn't.

Does anyone have any idea on what might be going on?

Thanks.

[phage434]

What is your plasmid origin? Low or medium copy plasmid origins can yield small amounts of DNA.

[Smog187]

My plasmid is a pHH2 vector, whose parental vector is pGEX-6. The ORI is pBR322

[phage434]

The pBR322 ori is relatively low copy, around 20, versus the 300-500 copy number for pUC19. This will result in lower plasmid DNA concentration.