I am trying to do PAGE of my pcr product. I could not get any bands in the gel and I used 10% gel prepared using TBE Buffer. Can anyone tell me what's wrong with my gel and what could I do?
Can you give some more details? Which buffers, running time, size of your product?
also - how did you stain gel after running (EtBr?) and how did you visualize bands (UV)?
How did you determine that you needed a 10% gel? That is very high.. 2% will resolve down to 50bp.
Did you connect your electrophoresis rig to the power source in the right polarity? That one bit me more than I'd like to admit in my undergrad days.
You use ethidium bromide at 5ug/ml either 1. you add to the agarose before it gels (but make sure it is not too hot), and then proceed to cast your gel; or 2. you add ethidium to the 1X buffer. In either case you must dispose of the ethidium containing gel or buffer in the appropriate way.
Use a 6% gel, run with TBE 1X or 0.5X at 70 V for 3 hour or more, 3 hour is right for 50 bp PCR, up to 6 hour for a 1000 bp you can increase voltage up to 100 V, decreasing the running time obviously. Use a DNA loading buffer and bromophenol blue run together 50-100 pb PCR. After, visualize with ethidium bromide.
remember adjust yur electrophoresis running time depending of PCR size.
greetings.
@Christian Praetorius its multiple bands (i am amplified using RAPD ) and i used TBE(tris ,Boric acid and 0.5 EDTA pH8) buffer , running time was 3 hr.
@Alejandro Martin yes the electrophoresis rig to the power source in the right polarity..i can see the dye running in the correct direction...
@Victoria Palau to determine the polymorphism i used 10% polyacrylamide gel...
@ Jose Villarreal Ascencio i amusing 0.5 X buffer and run in 50 to 60 V but not get even markers(100bp) in the gel...
use a DNA ladder for a positive control, you MUST see anything if you run the gel using conditions that i mentioned before. If not, recheck in an agarose gel that effectively do you have any DNA.
Hi,
Please clarify to some that you're running DNA by polyacrylamide gel electrophoresis (PAGE). Some are giving advice for agarose DNA gels which is not useful here. That being said, I've found PAGE DNA gels more challenging to stain than agarose gels. I found that PAGE gels absorbed EtBR fairly intensively and that destaining in TBE was needed to see bands well. Even under ideal staining conditions signal to noise can be less than ideal. Images provided by those specialized in making PAGE TBE gels show some transilluminator background in images http://www.precastgels.com/tbe_gels_for_dna_separation.htm
If you could find someone with a fluorescent laser bed scanner like a Typhoon-Trio, you'd be in better shape for imaging DNA bands in PAGE format.
Positive control DNA standard samples are good suggestions too.
How you are staining your gel, you can silver stain your gel to visualize the product. Compared to agarose it will resolve many pcr bands and the bands will be sharp too. Always load the ladder in the gel, it will act as a positive control for staining as well as give you the idea of the band size.
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