I have a couple of questions regarding DNA or RNA extraction in the laboratory.

1. Why is the DNA or RNA sample diluted in water or in a buffer to 1ml prior to putting the sample in the spectrophometer?

2. In spectrophotometric conversions for nucleic acids:
For dsDNA, an A260 of 1 = 50 µg/ml
For ss oligonucleotides, an A260 of 1 = 33 µg/ml
For ssRNA, an A260 of 1 = 40 µg/ml
My question is: are these values (55, 33 and 40 µg/ml) standard, i mean, the same at all times for Absorbance at 260 nm?
And what is meant by "Absorbance 260 of 1" What is the meaning of "1" ?

Question

I have a couple of questions regarding DNA or RNA extraction in the laboratory.

1. Why is the DNA or RNA sample diluted in water or in a buffer to 1ml prior to putting the sample in the spectrophometer?

2. In spectrophotometric conversions for nucleic acids:
For dsDNA, an A260 of 1 = 50 µg/ml
For ss oligonucleotides, an A260 of 1 = 33 µg/ml
For ssRNA, an  A260 of 1 = 40 µg/ml
My question is: are these values (55, 33 and 40 µg/ml) standard, i mean, the same at all times for Absorbance at 260 nm?
And what is meant by "Absorbance 260 of 1" What is the meaning of "1" ?

anonymous · Answer

@aaronq Hi. I need ur help please =)

shrutipande9 · Answer

we dilute it because u need to add a specific volume of sample in spectro....and obv d dna sample collected is not that much...so we dilute it...

shrutipande9 · Answer

these values are approximations..and not defined values...
if u check d absorbance at A260 and get d reading as 1....then d conc of ds DNA is 50microg/ml so "abosrbance of 1" is at particular wavelength if u get d absorbance as 1 then the given value is d conc of ur sample...:)

anonymous · Answer

you mean to say if the machine gives me a value of 1 when i use absorbance at 260 for dsDNA, ssoligo., ssRNA then the concentration will be 50 microg/ml, 33 and 40 respectively, this i got it.... but are these 3 values obligatorily (even if its an approx) the same all the times for A260 (absorbance reading 1)???

shrutipande9 · Answer

yes....see at absorbance 1 the cocn differs....

shrutipande9 · Answer

see consider this...u put dna sample...take conc at A260 and get d reading 1...then conc is 50 then for rna its 33 and so on...

shrutipande9 · Answer

*conc

shrutipande9 · Answer

u getting my point right?

anonymous · Answer

Of course i got it, thanks Shruti =)... one more question

shrutipande9 · Answer

sure...

anonymous · Answer

if you have to check the purity of nucleic acids in a given sample, you gotta measure its absorbance at A260 and A280 ryt? What's the effect? i mean how do i know if my sample is pure or not?

shrutipande9 · Answer

take d ratio of A260/A280
if the ratio is 1.8 exact then u have only dna.....if below 1.8 then u have protein impurities
if above 2.0 it has rna also

shrutipande9 · Answer

there is explanation for d values also...but i cant remember it right now....sry

anonymous · Answer

Thank you shruti, i appreciate very much. If u got to know the reasons, please tell me, its important for me.