pulsed field gel electrophoresis

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limin
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pulsed field gel electrophoresis

Post by limin » Sat Jan 13, 2007 12:56 pm

any one interested in PFGE??
any experts in PFGE? I need HELP!!
I keep geting smearing for my PFGE results.
and degraded DNA....

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canalon
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Post by canalon » Sat Jan 13, 2007 1:25 pm

Far from an expert. But have tried my hands at it. Usually smears come from DNA preparation.
Patrick

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Post by limin » Sat Jan 13, 2007 4:54 pm

thanks...patrick
got any suggestion to avoid it?will it affect the result of RE? I mean after digestion...
and do smear mean the DNA degraded?
i need to repeat doing plug?
or got any solution to solve it or improved?
before run d gel with digesion i run undigested before,d DNA CONTENTS IS enough,but got smears.after that i did digestion,the DNA degraded..what cause this?
and what r d precaution that i need to aware when doing plug?
hope can get your reply soon..thanks..

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canalon
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Post by canalon » Sat Jan 13, 2007 5:18 pm

If your DNA is degraded, you cannoit repare it. You have to redo the plug.

When you prepare your plugs always be gentle but as far as I remember, except for conditions of extractions that might be too harsh, or not strong enough (some bacteria have capsuls or other cell walls that make harder to use the regular protocol) there are no special precautions if you start with freshly harvested bacteria and you have prepared your plugs correctly.
Patrick

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Post by limin » Sat Jan 13, 2007 5:34 pm

will it due to the mixing of protinase K and agarose when doing plug?
coz my supervisor told me maybe it will cause burst of cell if the mixing not done by gently...then we should use 1ml pipette to avoid bursting of cell?
then how about over digested? what result will i get if over digested?

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canalon
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Post by canalon » Sat Jan 13, 2007 10:54 pm

You have to be gentle with your bacteria if you do not want them to burst. But As far as I remember you first make your plug, then you start dissolving cll walls and membranes. Since bacteria are quite resistant when they have a complete cell wall you do not have to be particularly gentle before this step.

You will get a smear too in case of non specific digestion.
Patrick

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Post by limin » Sun Jan 14, 2007 1:48 am

Image

This is my undigestion result, is it my plug preparation not good?That's y causing smearing?and the plasmid appearing, is it a common phenomenon?



Image


This is digestion result, the three wells with clear bands is markers.The others is my samples. using different enzymes.Marker by Xba1, whereas samples by Apa1.is it the Apa1 enzymes problem or my plug problem?

i suspect the Apa1 enzymes expired, but i have not enuf evidence, and it may cause by my plugs also right?

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Post by sachin » Sun Jan 14, 2007 4:55 am

I ve seen this first time.......

really good results.......
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Post by carton » Mon Jan 22, 2007 1:52 pm

Well, I think it is becuz of your enzyme, they may contain DNase.
most enzyme are extracted crudely from bacteria, especially REs
I met same situation while constructing a plasmid.


hope this will help :P

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Post by limin » Mon Jan 22, 2007 3:51 pm

thanks a lot ya...
i suspect d enzyme got problem also...

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