too confused about plasmid concentration? - (Oct/26/2006 )

hi there
i am actually confused with the plasmid concentration calculation. i isolated plasmid from a 200 ml culture and i resuspeded the final plasmid in water and i need to calculate the concentration , i did it by UV spec and got an OD of 1.7 ( should be between 0.1 -1.0) at A260,,, did with 1/100 dilution in water.. please do help.

-nish-

QUOTE (nish @ Oct 27 2006, 08:29 AM)
hi there
i am actually confused with the plasmid concentration calculation. i isolated plasmid from a 200 ml culture and i resuspeded the final plasmid in water and i need to calculate the concentration , i did it by UV spec and got an OD of 1.7 ( should be between 0.1 -1.0) at A260,,, did with 1/100 dilution in water.. please do help.

Applying the Lambert-Beer law, you get that 1mg/ml (or 1ug/ul) of DNA reads 20 OD at 260nm in a standard 1cm cuvette. You got 1.7 and your dilution factor is 100, therefore your undiluted solution would read 170 OD.
1ug/ul:20 OD=x ug/ul:170 OD

x= 8.5 ug/ul

-dnafactory-

QUOTE (dnafactory @ Oct 27 2006, 01:50 AM)
Applying the Lambert-Beer law, you get that 1mg/ml (or 1ug/ul) of DNA reads 20 OD at 260nm in a standard 1cm cuvette. You got 1.7 and your dilution factor is 100, therefore your undiluted solution would read 170 OD.
1ug/ul:20 OD=x ug/ul:170 OD

x= 8.5 ug/ul

that seems like a lot of DNA.

-scolix-

You may want to do another dilution 1:100 and verify that this result is real. Also, you should probably be diluting your DNA in TE buffer instead of water. It will be much more stable.

-tap14-

I would like to remind you (I'm sure you know it) to add the same amount of buffer that you have used to resuspend your DNA to the blank. So, if you added 10ul DNA to 990ul water, you should add 10ul buffer to 990ul water for the blank

-dnafactory-