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Electrophoresis problem , please help pals !


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8 replies to this topic

#1 M_shabani_medbiotech

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Posted 03 February 2013 - 08:08 AM

hi every one ...
i have this problem with my electrophoresis that the pcr product stays in the well even 1 hour after running has been started . does any one knows the reason ? plz help ! i need to extract the pure pcr product from the gel . but when all i load , stays in the well , how can i extract ?!!!

#2 Tabaluga

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Posted 03 February 2013 - 08:27 AM

Unfortunately I don't know myself, but I think there are quite some people with this problem...
There are lots of discussions and suggestions all over the web: https://www.google.d...iw=1024&bih=497

For a start you could read some of these, till maybe someone comes along who can give further advice...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 M_shabani_medbiotech

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Posted 03 February 2013 - 08:37 AM

thank you Enthusiast for ur answer :)

i should add that the size of my pcr product is 960 bp , so it is not that long to get stuck in the well :(
and besides , ladder runs almost properly but not my pcr product :(

#4 hobglobin

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Posted 03 February 2013 - 09:05 AM

First I'd check if the electrophoresis is really running, i.e. check for tiny bubbles at the electrodes when it's switched on. If this is okay the gel-percentage you also should check. Which percentage do you use?

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#5 Tabaluga

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Posted 03 February 2013 - 09:18 AM

What do you mean by saying the ladder runs almost properly ? Is it normal or not ? If it's normal the overall electrophoresis should be working, shouldn't it ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 M_shabani_medbiotech

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Posted 03 February 2013 - 10:28 AM

dear hobglobin , the electrophoresis is actually running , cuz i told u the ladder runs properly . and i use 1% agarose gel .

dear Tabaluga , i mean the ladder runs but the bands are not very sharp . i dont know what to do :(

#7 hobglobin

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Posted 03 February 2013 - 10:42 AM

over-read this, sorry. Other common problems with these description are overloading the wells and protein contaminations, but the latter would be surprising with a PCR sample.
Sharper bands you can get e.g. if you start with 80 V until the loading dye is in the agarose and then increasing to 120 V (and if TAE buffer I wouldn't increase more), fast loading of all the wells (avoiding diffusion), a sufficiently long polymerisation time of the gel. And running the gel not too long, as then diffusion becomes a factor.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#8 DrRupesh Kumar Rajput

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Posted 04 February 2013 - 04:20 AM

Hello Friend
In this problem with gel electrophoresis, you need to check the gel apparatus and current flow in the assambly.
Second thing is your running buffer and pH of the buffer.
You need to prapare new TAE buffer with apropriate pH and then run your product,
Load small amount of Dye in a wel, without sample which will be a marker or control,
Hope your product will run in a proper way and resolve with marker.
chears

#9 phage434

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Posted 04 February 2013 - 06:32 AM

Just to make sure. A common failure is to make the agarose gel with water instead of buffer. This leads to all sorts of difficult to interpret problems.




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