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Dye does not separe in acrylamide gel

acrylamide electrophoresis DNA

Best Answer mdfenko, 06 December 2016 - 05:29 AM

are you sure that there was sufficient crosslinker in the acrylamide, maybe it was prepared wrong? are you sure you don't normally use 19:1 (that's what i used for sequencing back in the day)?

 

check the pH of the running buffer, if it's significantly off then migration will be affected.

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#1 piemmea

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Posted 06 December 2016 - 04:18 AM

Hi all,

 

hope you have some clue. I prepared a 5% acrylamide gel for DNA separation using s(at least I think!) same reagents as usual: acryl. 29:1, 10% aps, etc. Gel polymerised, but when I loaded the dye (standard) for the prerun to clean the gel, I saw that Bromophenol blue and Xylene cyanol co-migrated and did not separe as usual!

 

I have no idea what happened, reagents were the same but acrylamide (not prepared by me, but at least it polymerised): do you have some?

 

tnx!



#2 mdfenko

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Posted 06 December 2016 - 05:29 AM   Best Answer

are you sure that there was sufficient crosslinker in the acrylamide, maybe it was prepared wrong? are you sure you don't normally use 19:1 (that's what i used for sequencing back in the day)?

 

check the pH of the running buffer, if it's significantly off then migration will be affected.


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#3 piemmea

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Posted 20 December 2016 - 02:33 PM

Hi, crsslinking worked as normal, I just re-prepared buffers and everything worked again. Tnx for the tip





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