Dear All
I want to calculate the Tm value of Salmon Sperm DNA, that I purchased from Sigma (31149-10G). The DNA come in powder form, that I was dissolve and purified by standard protocol. The Abs is given A230 = 0.830, A260 = 1.898, A280 = 1.041, A340 = 0.004 (A260/280= 1.83, A260/230= 2.29).
Then that DNA I dissolve in 10 mM sodium phosphate Buffer and make final conc 30 ug/ml of DNA for Tm calculation. But I am getting this types of plot as attached in picture.
Please suggest me to come out this problem.
Thank you in advance!
I am not sure that your DNA is correctly dissolved. Dissolving genomic DNA from lyophilized powder is a very long process .... several hours at 60°C. Secondly, your source DNA has a high protein contamination (around 5 %) which will cause a lot of noise in your absorbance plots.
As Shaheed Reza noted, Tm calculations are entirely inappropriate here. You can generate melt curves (and thus melt peaks) for relatively short DNA fragments that are all the exact same sequence (such as the product of a PCR), because short fragments of identical sequence DNA will melt relatively homogenously. Note, however, that the melt temperature increases with length, and with GC content, and is asymptotic: 95 degrees is hot enough to melt just about any DNA.
Thus, melt curves will readily distinguish between a 40bp primer dimer product and a 400bp PCR product, but will be unlikely to provide any useful distinction between a 400bp PCR product and a 440bp PCR product.
For genomic DNA, you have several billion bases per genome, and the sequences range from highly repetitive and simple, to highly repetitive and complex, to homologous regions, to unique regions: the melt curve will be, in effect, a mess. Which is basically what you're observing.
Interestingly, you can see (if you're careful) these sequence elements reassociating, if you cool denatured DNA slowly:
http://en.wikipedia.org/wiki/Cot_analysis
So a pertinent question at this point would be: why are you trying to determine the Tm of salmon sperm DNA?
Ian Robbins Sir ...The DNA was dissolve in MQ water for several hour and then this DNA was separated from Protein with standard protocol (Sigma supplier DNA has 5% protein contamination). So how you know this DNA contain 5% protein contamination? Is my protocol is wrong and I am not able to remove all protein in my sample.
John Hildyard sir ...... I want to study the effect of solvent on the melting temperature DNA. So as model DNA I was taken this salmon sperm DNA for my study.
Hrmmm. It might be more appropriate to start on a smaller scale with fewer variables, then.
Take a 200bp PCR product (I'd recommend making a fair bit), purify it to homogeneity (gel electrophoresis followed by gel extraction, for instance), then run melt curves for a fixed amount of this product + increasing concentration of solvent. If your solvent is lowering the melting point, you should see a progressive leftward shift (to lower temp) and probably progressive broadening of the derivative peak.
As the other respondents have pointed out it will be difficult/impossible to generated meaningful data from a melt curve analyses of what is essentially a whole genome. ON thinking about this it reminds of the types of hybridization experiments people use to do way back when trying to determine the genetic relationships between species. In these experiments they would label one genome completely denature and then try to hybridize it to another genome which was immobilized (dot or Southern blot). Initial hybridization was done under low stringency - usually high salt, then, the stringency was increase by reducing buffer/salt concentrations and increasing the temperature.You might be able to do something similar with a designated set of parameters (salt/temp) then add in your solvent and determine the effects on hybridization. Not modern or eloquent but it might work!
From what you have described previously about the aim of your study - to determine how solvent concentration affects the melting profile of DNA. As John H has described would best be done with a small fragment of DNA such a PCR product. If you want to see at the genome level you need to be able quantify or estimate the ratio of ddDNA to ssDNA - you might be able to use a technique such as RAPD PCR where you use non-specific primers, if you were to use one set of annealing conditions as a reference point, then add in your solvent and keep everything else equal, the solvent might increase access of the primes to the template (ie ssDNA) and give you different profiles.
See - http://www.ncbi.nlm.nih.gov/probe/docs/techrapd/ The difficulty with RAPD can be keeping your standards consistent and you'd need to devise a system to score the output. And also carefully think about and detail the underlying assumptions!
From what seems like a simple initial question some complex solutions.
- Badges
- Science method