Frowning of samples usually means overloading of the dna sample which should be 50-100ng per well but it is also worth checking that the gel is completely polymerised and that the wells are thoroughly washed with buffer just before sample loading.

Dear Paul,

Thank you so much for your answer! Letting it polymerize longer and washing the wells definitely helped. We changed following things:

• letting gel polymerize 90 minutes instead of 60.
• using about 8 ng extracted DNA before PCR instead of 3 ng DNA
• washing wells with water ones and with TAE buffer via pipetting up and down
• using smaller wells
• starting run with 5 min at 200V, then 4 hours at 130V (would you recommend longer running time at lower volts?)
• trying 50% diluted (red in image) DNA and cleaned DNA (ethanol precipitation, blue in image)

Our changes for next run:

• cleaning glass with different soup, rinsing in buffer, drying
• preparing fresh TAE buffer and APS solutions

Do you have any other recommendations based on the image?

Felix and Jonas

Well done Felix Biefel things are improving. am still worried about the dna ladder and the bottoms of the samples are still looking ragged and the ladder smeared. I would give the gel longer to polymerise because I believe that the most likely cause is old APS so definitely try new APS ( I never used dissolved APS more than 3 days old. I am thinking that the ladder runs badly because it is next to the edge of the gel and old APS maybe combined with oxygen from a loosely fitted gel frame may cause the gel to polymrerise badly. Try a slightly lower running voltage but if the new APS works well then you can reduce the gel setting time in the future

Good luck,

Paul

Dear Paul Rutland ,

Thank you so much for your last answer! We conducted additional gel runs, but unfortunately, the results have not improved significantly. We extended the polymerization time to 2 hours, used fresh TAE buffer, and ran the gels for 16 hours at 50V. We also tested different gradient conditions (30–50% and 0–30%), yet we are still seeing unclear bands and smearing (see attached images).

At this point, we’re running out of options. We’ve changed multiple variables, but the smearing persists across all runs, suggesting that a common factor might be responsible. One possibility could be the urea, as it's relatively old, while all other reagents are fresh?

Do you have any suggestions on how we could further optimize the process? Your insights would be greatly appreciated!

Best,

Felix

Hello Felix Biefel , This is disappointing and I can only think of 3 reagent based areas that can spoil sample running. 1 APS absolutely must be freshly made.....try borrowing a new ,younger bottle from another lab.

2 Formamide can become acidic and degrade dna samples. Either find a newer fresh bottle of formamide or add a mixed bed resin to the formamide you are using so that all acidic species are removed from the formamide

3 It may be that your acrylamide is getting old....again find a different source of acrylamide and Bis from another lab

good luck

Paul