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SDS-PAGE Gel Electrophoresis Forum SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


Sds page voltage current

Sds page voltage current - SDS-PAGE Gel Electrophoresis Forum

Sds page voltage current - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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  #1  
Old 11-18-2010, 05:51 AM
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Default Sds page voltage current



Dear All,

I m running minigel of 5%stack and 12%Resolv gel, with TRIS HCl buffer

with voltage 100 for Stack and 150 for Resolving with 21mA constant voltage

but the tracking dye is moving unevenly, and Bands are also not visible properly sometime no bands - check the attached Gel Pix



Do suggest to improve my technique

regards

Arun
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Last edited by admin; 11-21-2010 at 09:48 PM.
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Old 11-18-2010, 05:07 PM
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Default Re: Sds page voltage current

Get new buffer, use less loading dye.
When you start electrophoresis, visually check that the bubbles created are 1) strong constant amount, 2) evenly distributed across the length of the apparatus.

Last troubleshooting tip: Check your protein concentration, depending on your extraction method, the higher the protein the higher the salts carried along, you can rid most of the salts by starting electrophoresis for 5min at higher voltage, like 140-150V (since you're currently doing 100V;which is low BTW), then restarting for the regular gel run at 100V.
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Old 11-19-2010, 12:08 PM
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Default Re: Sds page voltage current

Many thanks.

I'll sure apply ur points, lets hope for Good.

Meanwhile I have taken pix of todays gel while running, which is just made up of Resolving gel of 10%, now the tracking dye is moving parallel Pix attached.

Also, My Power pack is giving me a max of 100 V during Stacking and a max of 160 V during Resolving, Current is kept constant 21mA---- is this is related to Running buffer problem or something with Power pack.



Suggest me...

Many thanks

Regards

Arun Kp
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Old 11-21-2010, 11:25 AM
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Default Re: Sds page voltage current

No need to run the gel on 2 different voltages; and most importantly do not use stored/precasted gels. Prepare fresh gel; we too had teh same problem in the lab and it has been solved.

I hope this will be helpful.
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Old 11-22-2010, 04:38 PM
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Default Re: Sds page voltage current

Thanks Alot,

I have used all the time a freshly prepared gel. is that ok if i run only 10% resolving gel without stcking...???
i even did that but didnt get any band not even in marker lane...

suggest pls...

Regards

Arun KP
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Old 11-22-2010, 04:41 PM
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Default Re: Sds page voltage current

Thanks Alot,

I have used all the time a freshly prepared gel. is that ok if i run only 10% resolving gel without stcking...???
i even did that but didnt get any band not even in marker lane...

suggest pls...

Regards

Arun KP
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Old 11-23-2010, 03:28 AM
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Cool Re: Sds page voltage current

Quote:
Originally Posted by kparunscientist View Post
Many thanks.


Meanwhile I have taken pix of todays gel while running, which is just made up of Resolving gel of 10%, now the tracking dye is moving parallel Pix attached.

Also, My Power pack is giving me a max of 100 V during Stacking and a max of 160 V during Resolving, Current is kept constant 21mA---- is this is related to Running buffer problem or something with Power pack.

Arun Kp
Keep voltage constant, not amps.
Amps are only kept constant for 2D electrophoresis (1st separation when voltage is 10,000V+) and semi-dry Western transfer.
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Old 11-23-2010, 01:19 PM
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Default Re: Sds page voltage current

I think you have some problem with buffer system or dye. see the gel you posted previously looks fine now the gel is worse. you do one thing just change everything and prepare buffer in autoclaved water. and use proper amount of dye. And refer sambook russel for electrophoresis. Its quite common technique why u r getting problem is out of my mind. If problem prevails the you r from India and even me too. you can call me on 8939069747

Bye
Shabir
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