Maxiprep Question - (Sep/21/2005 )
I would not trust a protein measurement by spec if you do not perform a bradford assay or somehting similar. As far as your two-layers go, I have seen this before after the elution and it is not a big deal. Go ahead with the isopropanol precipitation/70% ETOH wash and your DNA should be good to go. The 260/280 measurement is the most meaningful assessment of your DNA purity in this case.
Thank you all for the suggestions...
The 2 layer maxiprep is a lentivirus DNA for transfection.....
This is a lentivirus DNA and I will add the packaging DNA and etoh ppt it before the transfection....
Do you think is it ok to do the etoh ppt after I adding the packaging DNA ...or is it better to etoh ppt the lentivirus DNA first then add the packaging DNA then etoh ppt it again ?
Follow he protocol and procede with the isopropanol precipitation, resuspend final DNA in TE. Calculate the concentration and use that for transfection. This is better because you know exacly how much DNA from each plasmid you are adding. I don't know why you would need to do another round of ETOH ppt. unless your DNA is very dilute.
thx for the reply....
Right now I still waiting for the cells to be ready for transfection...
I recalculate lentivirus dna concentration from the maxiprep again and at this point, the double layer is gone I don't know why but it just gone and become 1 layer....
dna concentration is 1250ug/mL and 260/280 ratio is 2.1 (I use silica based column for maxiprep)
when I calculate it again the spectrometer always give the different number of DNA concentration...
I just bit confused about why I got 260/280 ratio of 2.1.....is anyone has a suggestion about this?
What are you using to blank the spec?
I use milliQ water..
I think that to know whether you have or not contamination of the DNA with high amount of protein you should consider the OD260/OD280 ratio.
If the ratio is 1.76 when measured in TE buffer then you might have 65-70% of protein contaminants. If you measured the OD in H2O, the real ratio might be higher therefore you have much less protein. You should measure in TE and use TE as blanck.
To know more about this you might look up the table I pasted in this topic, reply from 20 Dez:
and if you want to know how you should dilute your sample in order to avoid high variability between measurements you can have a look to the following topic also from 20 Dez:
One thing I don´t understand. How did you measured the protein concentration?
To measure protein concentration you need a protein like BSA for eg. with know concentration and use that as to make the curve. Only after you can estimate the concentration of protein in your sample.
Which absorbance did you use to measure protein concentration?