Need to work out how much to dilute samples to get same absorbance values - (Dec/02/2011 )
Hi there, really hoping that someone can help as I'm getting all muddled! Basically I have re-suspended different pelleted e.coli cultures (containing different plasmids) in 1 ml PBS (or in the case of 1 of them only 0.5 ml PBS as there weren't many cells). I was then told to measure the absorbance of each solution containing 50 ul of solution described + 950 ul PBS (20 x dilution). The aim is to get each of the solutions to the same absorbance for use in a western blot . So the question is, given these absorbance values how do I calculate how much PBS to add to the original solutions to get them to be all the same absorbance? I know this is just maths but I'm stuck. To give an example, I need to get to an abs (at 600nm) of 0.377 for each and the readings are e.g 1.534 (that's 50 ul of 1 ml ecoli suspension and 950 ul PBS) sorry if I've repeated myself - just trying to be clear. Hope that someone can help so I can do my western!
Absorbance should be directly proportional to concentration. So try to dilute it 1.534/0.377 x times and see if it is true. But as I recall only certain range of absorbance can be trusted, absorbance higher than ~1-1.5A is out of valid range for instrumental reasons (you should check the manual). In that case first dilute it a bit and then measure again.