Factoring dilutions into calculations

[sven222]

Hey guys, gals (Monique )

Quick question. I recently completed a labortatory session in my Genetics class regarding the isolation and analysis of plasmid DNA, and a section of the report has me stumped.

Before the plasmid DNA was put under the spectrophotometer, it was diluted in 285µl of distilled water (15µl of DNA, all up 300µl). Absorbance was measured, and found to be around 0.2 (Don't have the exact figures on me). Now assuming A=1 is the equivelant of 50µg/ml, we need to calculate the concentration of the plasmid DNA sample, taking into consideration the dilution.

I realize this is simple maths, but for some reason when it comes to facotring dilutions into my calculations, my brain laughs at me and tells me where to go. So I need your help guys :)

Thanks in advance.

 

[iansmith]

Your dilution factor is 20 because 15 in 300 give you a 5*10-2 dilution which is 1/20.

The formula to calculate DNA concentration is usually as follow

(Net A260)*(dilution factor)*(DNA constance)

Net A260=(Blank A260)-(sample A260)

 

[sven222]

Thanks for the reply iansmith, greatly appreciated.

Firstly, wouldn't the net A260 be sample - blank? Rather than blank - sample?

And when you say DNA constance, I assume you're referring to the 50ug/ml I mentioned above.

Besides that, I didn't realize it was that simple. It must be the lack of sleep, or the week old food I've been eating.

Thanks a heap

 

[iansmith]

Originally posted by sven222
Firstly, wouldn't the net A260 be sample - blank? Rather than blank - sample?

my mistake it is sample-blank

Originally posted by sven222
And when you say DNA constance, I assume you're referring to the 50ug/ml I mentioned above.

yes it is