DNA size calibration?

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cloudnine
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DNA size calibration?

Post by cloudnine » Fri Mar 24, 2006 3:06 pm

hello all,

i have some problems in calculating the size of plasmid DNA after electrophoresis.
Lambda Hind III DNA was used as marker.

the standard calibration curve of the marker was drawn on semi-log graph paper.
the sizes of extracted plasmids were calculated by comparing relative mobility of the plasmids and marker, and then using the calibration curve, the sizes were estimated.

BUT, i have heard recently that in the analysis of circular DNA (i think most of plasmids are circular)
is that right?

if it is so, is there another possible ways to find such results?
we don't have circular DNA so we are using Lambda os mp

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Post by canalon » Fri Mar 24, 2006 3:41 pm

Arrgh what have you heard aboutcircular DNA?

What I can tell you about it is that it doesn"t move the same way depending on super coiling. And since it is rather hard to know the degree of supercoiling of a plasmid in advance and using equivaletly supercoiled marker, it is best to linearize your plasmid (single cut restriction, or if not single cut, additions of sizes).
Patrick

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any proof. (Ashley Montague)

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Post by cloudnine » Wed Mar 29, 2006 3:47 am

thank you for your reply and suggestion

i hate to say that we don't have enzymes to make restriction.
so that i wonder whether there would be some way for the estimation of size.
as you have said, i am little bit dissapointed.
anyway, thank you for your suggestion.

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Post by canalon » Wed Mar 29, 2006 4:22 am

There are probably some way to cut DNA non specifically with some chemicals, but I have to admit that I never was in a situation where I couldn't put my hands on some restriction enzyme.

Nevertheless your measure maybe accurate, it's simply impossible to be sure. I remember that some plasmids supercoiling were changing with the salinity of the medium, and comparing cultures at different concentration of salt where you could see the different configurations (depending on the number of coiling. If I remember correctly the largest size was the uncoiled plasmid (less compact structure).

I am not sure, but if you could transform your bacteria with a plasmid of known weight, it could be extracted ans used as a standard, assuming the degree of supercoioling would be identical (that is the wild guess, with the ability to transform your strain).

Good luck
Patrick

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Post by cloudnine » Wed Mar 29, 2006 4:34 am

i am about to sumit my thesis to the University.

with this problems, i have some difficulties to express my results.
should i express my results calculated with linear Lambda HindIII (linear) marker? is that acceptable?

i am afraid to say that our lab is now in the first steps in Molecular Biology and not equipped enough and so we can't afford to do some later works of research.
if it is not acceptable results, should i say something in 'discussion'?

i hope you understand my situation and give suggestion to me.

thank you, really.

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Post by canalon » Wed Mar 29, 2006 4:42 am

EcoRI is cheap, sold with its buffer and do not require anything else than a water bath or a 37°C incubator to digest DNA. And you can load your digestion directly on a gel.

It is not the only enzyme, and you could probably borrow a few microliter of enzyme and some buffer from another lab better equiped. If you already have run some gels you can guesstimate the quantity of DNA by the intensity of fluorescence (which directly depends on the number of base pairs in the DNA molecule), and hence be sure to have a complete digestion of your DNA when loading it on gel.

Otherwise you could have a few words abou it in your discussion. At least it would show that you understand what you are doing and the limitations of your work
Patrick

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Post by cloudnine » Wed Mar 29, 2006 4:58 am

as molecular biology is not so popular in our country, i have some difficulties for some help.
although i don't have much time, i will try my best before the submission.
thank you very much.

PS. i have encounterd some problems during gel electrophoresis of some gels.

the bromophenol blue dye turned into yellow and one-third of the gel was damaged. ( no bands appeared even markers)

voltage was 50 V and running time would be around 1 hr.
this occured in about 40-45 minutes, i think

i think the pH changes may be the reason, and the buffer (TAE) was so hot after electrophoresis.

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Post by canalon » Wed Mar 29, 2006 5:08 am

Yep color change is due to pH change. TAE or TBE are always warm after electrophoresis, except that at 50V it is not that warm Of course it also depends on the size of your gel. Running your gel at 5V/cm of gel is really safe, you can go up to 15V/cm if you are in a real hurry and precision of the band is not of the essence, but then the buffer runs really hot and the gel starts melting....
Ar you sure of the concntration of the ingredients of your buffer? because that could be a reason.
Patrick

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Post by cloudnine » Wed Mar 29, 2006 5:19 am

i think at that time, the buffer stock solution was about to empty.
and the buffer (diluted to 1x) in the gel tank was used for 2 consecutive runs of electrophoresis.
as you said, that might be the reason.
my sincere thanks for your time.

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