I've just begun learning DNA purification from whole blood- we are using the Qiagen Flexigene kit, and the protocol is very straightforward. However, when I attempt to quantify the concentration using the spectrophotometer (Ultrospec 2100 pro) I get wildly different numbers depending on the dilution (dilution factor 16, 44.3 ug/ml; df 8, 39.5; df 4, 47.1). Is this just a result of improper pipetting technique, or can this much variability be expected? Also, using the kit, the purified pellet is resuspended in 10mM Tris-Cl; I've diluted the DNA for spectrophotometry in both Te and water, and when I use Te, the absorbance at 230 is negative. Does this just result from having a lower EDTA concentration after diluting the DNA? Also, should I actually be diluting and blanking just in Tris-Cl and not Te? The lab assistant told me to use Te, but she said that she does not usually do the purification, and the doctoral student who had been doing the protocol is long gone. Also, the 260:280 ratio, no matter what the dilution type or factor, is always within a couple hundredths of 1.8.
Sorry if these questions are obvious. I'm in an epidemiology program, and I am just learning the basic assays necessary to analyze my samples- I am not a molecular biologist by any stretch of the imagination. I definitely need reliable estimates of concentration because the samples will be used for global methylation analysis, and I'll be diluting the samples to a concentration of 25ug/ml (we quantify using Picogreen when doing the assay, but we need an approximate of the concentration beforehand to make the stock)
Thank you! Any help would be greatly appreciated.