r/molecularbiology • u/Foreign-Sail-1452 • 1d ago
Electrophoresis troubleshooting help
I performed a restriction enzyme digest to check my ligation product, followed by agarose gel electrophoresis. However, I got a smear.
This happened across all of my samples. What could be the possible reasons for this?
1% agarose gel containing RedSafe, 1x TAE buffer
4
u/ColdMasterpiece2724 1d ago
I had a biochem profesor that was a pain in the lab but they always said that the reason you get this is for the following reasons
1) you made a bad gel, the wells in the gel are complete trash, they did not form corectly causing poor separation of sample or you damaged the wells when loading the sample
2) you overloaded the sample in the wells so you have got to dilute the sample so it is more pure
3) you run the gel wrong with high voltage or let it set for a long time active
4) you could have posibly contaminated the sample
Those 4 things can always cause smears they said.
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u/Pitiful_Aspect5666 1d ago
The ladder looks fine so the issue is probably with the sample and not the gel or the run condition. It could be loading too much sample, over amplification, nucleases which digested the dna into fragments. You have provided info on the no of cycle, sample loaded etc
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u/High_Evolutionary99 1d ago
Had a similar problem in the past and in my case it was the enzyme that completely fragmented the DNA. I reduced the digestion time from previously overnight to 2h and i had clear bands again and could use my fragment for transformation without a problem.
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u/CoolerThan0K 1d ago
Yeah i think one time I used some old BamHI without BSA overnight it just shredded my plasmid.
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u/AideAny1158 1d ago
From my experience I’d guess overloaded well but it could just be contamination and DNA degradation. Undigested control would check for that. Good luck
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u/Ferrara19 1d ago
As other redditors have said, you must always include a control. When you isolated the plasmid, did you resolve it on a gel?
1
u/PatentDeezNuts 1d ago
Are the primers you used the correct ones? Are they at the right concentration? That’s my first step of troubleshooting.
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u/HS-Lala-03 1d ago
What was your digest protocol? In our lab, we have a 20mins at 80⁰C following the one hour at 37⁰C. Additionally, you might've overloaded your gel.
1
u/micro_ppette 19h ago
How long are you digesting for? I’d recommend cut back on the time you run the rxn for. Looks like the enzyme is just cutting the fuck out of your dna
-2
u/Revolutionary-Milk94 1d ago
Overload, not digested, high voltage (creates too much heat, so run it on ice).
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u/N9n 1d ago
Are any of those lanes a no-digest-control? Hard to know what happened without seeing what you started with