The DNA polymerase gene from Bacillus licheniformis is over expressed and purified. I need to calculate the activity of that protein but that DNA polymerase is stable only upto 60 C therefore the PCR methodology can not be used. Can i know another method to detect activity?
,may be a polymerase from other source(e.g. Archaia) can do the job.Or what about
directly measuring DNA concentration spectroscopically (this might work or not depending on instrument sensitivity and expected amount of DNA produced)
Here is a non-radioactive method I published that uses fluorescence.
https://www.ncbi.nlm.nih.gov/pubmed/16266678
very nice work Adam.I am a theoretician but I thought something like that existed.However there is also an interesting theoretical problem there.As far as I know
most people use the r-6 Foerster dependence for the transfer but this should change in membranes,salt solutions etc because what matters is actually the
Green's function dyadic which is difficult to calculate broperly except for the bulk case
what do you think about this.?
I am unfamiliar with the term "Green's function dyadic." Are you referring to the orientation factor kappa-squared in the Forster theory? This parameter is generally assumed to have the value 2/3, but this assumption could be inadequate in some environments. There is a method for putting boundary values on kappa-squared (Dale, R. E., Eisinger, J. & Blumberg, W. E. (1979) The orientational freedom of molecular probes: the orientation factor in intramolecular energy transfer, Biophys.J.26,161-194) that I once employed when using a membrane-based FRET system (http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1997.00122.x/pdf), but it yielded a wide range of values.
Dear Adam
Our discussion reminds me of things I was thinking about when i as in Goettingen
20 years ago and could not finish them due to adverse circumstances.The factor
you mentioned is a very crude way to account for the orientational averaging of
the dipole-dipole interaction which enters via Fermi golden rule the matrix elements
defining the Foerster FRET mechanism. What I am addressing is the fact
that everybody uses a dipol-dipol interaction form with r-3 distance dependence which arises from differentiating The bulk Green's function
which is the potential of a unit point charge at r at a different poitnt x' equal to
the coulomb form only when all boundaries are infinitely far away..This is clearly
not the case with macromolecules ,,membranes etc.One has to treat the boundary
conditions (geometry,dielectrics,salt screening etc) in constructind a better
Green 'function G from which forming the gradients with respect to r and r' one
gets a tensor with components( Dx Dx'G etc) one gets a more correct distance
and angular dependence of the Foerster coupling in the complex systems
we discuss.This tensor is the dyadic green's function I meant.
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