DNA extraction and blue dextran as a DNA carrier - (Aug/22/2011 )
when you use blue dextran as a DNA carrier for DNA extraction, the dye is precipitated with DNA as a blue color pellet. when you measure the quantity of DNA, ( DNA in TE buffer), how to avoid the error as blue colour to the solution is going to be there while DNA is measured using nanodrop. I used 0.1% blue dextran in the begining with cell lysis buffer and extracted with phenol chloroform and acetate precipitaed the DNA. Anybody used blue dextran for this purpose and any advise?
At which wavelengths is dextran blue absorbing light?
You should add the same concentration of dextran blue to the blank.
You are right. That is what I was thinking of doing , but then I started with 0.1% dextran of 100ul added to lysis buffer of 200 ul and then went on added phenol chlor extraction and precipiatted with sodium acetate. So probably I may have to add 0.1% 100ul dextran in 200ul TE to make to the same dilution to measure right ? I can calculate the dilution here and make it to the same. I think dextarn as a sugar doesn't absorb the light it seems. Like glycogen, it doesn't affect the measurement, but blue colour , I am sure will affect. However I appreiciate your idea and I think that is the only way we could do this. I know that PCR amplification will not get affected by dextran. So it is the measurement which will be faulty. Thank you.
But the blue colour is due to chromophores in the dextran molecule itself, there's no extra blue colour added. So you cannot be sure how much of the dextran you precipitated together with the DNA and lost before in the lysis steps.
The absorption maximum of dextran is around 500-700 nm, so perhaps at the uv range for DNA it's negligible. I'd try out by measuring the absorbance of a dextran solution of appropriate concentration (or different possible concentrations) against a dextran free blank at the wavelengths you use for DNA. If there's nothing you can ignore it, if there's absorbance, you can IMO only estimate the dextran amount left.
Great advise. thank you.