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DNA SDS-PAGE electrophoresis

DNA SDS-PAGE INSERTION

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#1 trilobite

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Posted 16 December 2014 - 12:42 PM

Hello guys,

we are studying a 5bp insertion and for this, we perform a PCR method followed by SDS-PAGE electrophoresis. The method has been validated and yet, this gel was given to me today. So far, I has been given gels having only a single band as the ones shown in lanes 3-4. This molecular weight corresponds to the wild type. Do you have any idea what the other could be? Could they be a heteroduplex? 

 

Regards,1.jpg



#2 mdfenko

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Posted 17 December 2014 - 07:22 AM

i'm not sure i understand what it is that you are doing. pcr will amplify dna, sds-page is for the separation of denatured proteins. if you put a pcr reaction on sds-page then you will see the polymerase.

 

would you give us more information so that we can properly evaluate your situation?


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#3 JDSBlueDevl

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Posted 10 January 2015 - 11:16 AM

I know that SDS-PAGE can be used for smaller DNA fragments (usually up to 300 bp).  However, mdfenko is right.  I don't know why you are using an SDS-PAGE gel when you can PCR with a primer specifically directed toward your 5-bp insert, perhaps in conjunction with a reverse primer that will allow for a decently sized amplicon, and run a higher-percentage agarose gel.  Are you using such a primer?



#4 trilobite

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Posted 11 March 2015 - 03:34 PM

Hey, there, apologies for the inconvenience (this is what is happening when you attend a forum being at a conference)

This is an 5% polyacryamide gel.

 

Since my first post, I have re-visited the PCR conditions (running a gradient PCR, etc)

These 2 gels are indicative running the same samples in polyacrylamide and agarose gels, respectively. The same ladder is used. In both cases, this is a "conventional" PCR, not and HDA 

 

:-/

 

Screen Shot 2015-03-12 at 1.30.55 AM.png

Screen Shot 2015-03-12 at 1.31.02 AM.png


Edited by trilobite, 11 March 2015 - 03:51 PM.


#5 mdfenko

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Posted 12 March 2015 - 04:05 AM

resolution with page is finer than with age. you may just be resolving a thick band.


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#6 trilobite

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Posted 12 March 2015 - 04:44 AM

thank you, mdfenko. nevertheless, if you take a look between the two photos, you will see that the mw of the bands differ (depending on the same ladder used in both cases-the ladder being compatible with both agarose and polyacrylamide gels). this is more what drives me crazy, as they are the same samples. 



#7 mdfenko

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Posted 12 March 2015 - 05:03 AM

have you identified the ladder bands?

 

the separation is different between page and age.

 

also, even with the same running buffer (are they the same, including dilution?), the differences in restrictiveness of the gels makes a significant difference in mobility.


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