Debate and discussion of any biological questions not pertaining to a particular topic.
7 posts • Page 1 of 1
I am new to this and I am trying to workout the base pair concentrations.
If I have a solution of 30 uM ethiudium bromide and I am adding 2 uM of 12 mer oligonucleotide in double strand, which is the concentration in base pairs? Is it simply 2 divided by 30?
Thanks a lot
I think the goal is a ratio of ethidium molecules to base pairs, likely to determine whether the oligo is saturated with dye. As Jack pointed out, you need to multiply the molar concentration of the oligo by the number of base pairs in the molecule to get the effective concentration of base pairs. Then you compare that number with the molar concentration of the EthBr. It appears the EthBr will be in slight molar excess.
Since EthBr is an intercalator, I would think this would more accurately predict saturation if you use the number of base pairs minus one. That way you are counting the available spaces between base pairs for intercalation.
I should point out that I've never seen this calculated before.
Guys thanks for your reply. It is just at beginning of my Ph.D. and I have not a clue on how to report this concentrations. I found a lot of papers talking about this base pair concentrations. Do you have an idea where I can read more about it? Do you have any link to suggest? Thanks a lot in any case
The other question is: why I have to multiply for the number of base pairs if I obtain the concentration of the oligo with UV-vis using an epsilon. The epsilon has already in count the number of bases since it is estimated with the nearest-neibour principle. Am I wrong?
Sorry I start to loose it
If you have a molar absorbance for the oligo, it converts the absorbance to the concentration of the oligo, which is a count of entire oligo molecules per volume. If you want a count of base pairs per volume, you will need to multiply by the number of base pairs in the oligo. The result will be in (moles base pairs)/l instead of (moles oligo)/l.
I assume that when you are talking about the estimate from nearest-neighbor principle, you are probably referring to a correction for base-stacking hypochromicity. If so, that is not a way of integrating the count of bases into the molar absorbance, it is instead a correction for a difference between the absorbtion expected if the oligo were digested into free bases and the lower absorbtion of the intact oligo due to pi-pi interactions as the bases stack. It's not a correction for the number of bases, but for a shift in the energy of the absorbtive transition due to orbital interactions (that it, it corrects for the proximity of the bases to other bases and their relative orientation).
The hypochromic effect is the decrease in absorbance due to stacking. Compare this with the hyperchromic effect on melting of DNA duplexes:
http://www.sciencedirect.com/science?_o ... archtype=a
7 posts • Page 1 of 1
Who is online
Users browsing this forum: No registered users and 8 guests