Hi, just looking for some help with this. I can't for the life of me figure out what's causing this banding pattern. All my bands are producing this smily face pattern and neither I nor the rest of my research group can work out why this is happening to just my gels.
This is 1% agerose, run at 120V for 45 mins (it's a small gel).
Similar banding at 100V.
Tried using new buffer.
Loading 12 um of sample with 2 um of purple loading dye.
The agerose and buffer are microwaved for 2:20 (for 100 ml).
3.5 um of gel red is then added after the microwaved solution is allowed to cool for a few minutes, when is is mixed in by swirling it around a few times, before pouring the gel.
Any help with this would be much appreciated as these gels cannot be presented for obvious reasons.
@Harry,
The gel is not heated evenly across the lanes. High voltage is frequently the culprit. To avoid this, you can gently run the gel (lower the voltage 100V) to lessen temperature inconsistency.
Another thing, I see you have experienced a dimer formation (the lower line with high intensity bands, aren't you?) This may be reulted from lower template concentration, high primer concentration or poor annealing conditions annealing temperature etc.
@Lahiru
Thanks for the response, we have tried 100v but had no luck with that so I think we will try 80v.
With the primer dimers that have been forming, it's a consistent issue we've been having as well. We are using the annealing temperature that we calculated, might try a gradient of temperatures to get the best result/best annealing temperature, see if that makes a difference. We have already diluted our primer concentration 10 fold from the stock concentration. We also added DMSO to our reaction to try and get better results. If you've got any other ideas on what we could do to stop dimers forming that would be amazing.
This effect often occurs when you add too much DNA in the wells. My suggestions would be, (1) add less DNA (about half of whatever you adding now) or wider well; (2) Voltage 80-90; (3) use fresh buffer
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