Extraction of genomic DNA from 10 fold serial dilution of cells. - (Jan/15/2007 )

hi all,

I am trying to do a comparison of cell numbers between viable count and quatitative real-time PCR. However i am facing problem in extracting DNA from 10¹, 10², 10³, 10⁴,10⁵,10⁶,10⁷ of cells/ml. I do a 10 fold serial dilution of the bacteria cells and use 1ml of the dilution to spin down to continue with lysozyme incubation. However, i cannot get any cell pellet and i guess it's because the number of cells is too little. What i am worry here is i might not get any DNA in the end of my extraction. Should i use bigger volume of the dilution? Please suggest what is the routine way to do it?

Thanks.

Regards,
Connice

Why spin down? Maybe resuspend your 10-7 pellet in the buffer (without SDS or lysozyme) and then dilute in more buffer. Then add the SDS and lysozyme on top and continue with your extraction. I think with centrifugation, you always lose about 10-2 cells so I would avoid doing that.

Oh ya..... why i never thought of that.... Thanks a lot. I will try to do it.

Regards,
Connice

May want to add a carrier DNA or glycogen when doing you precipitation to prevent differences in prec. efficiency at lower DNA concentration ranges.

I am not sure about the linear range of DNA isolation efficiency with qiagen columns. I was referring to carrier DNA if you do a EtOH prec.