I've been doing molecular cloning for many years now and I've recently moved labs. In my new lab I'm having an issue with band splitting in my DNA gels, the lower bands in particular look letterbox shaped (see the smaller fragments of the DNA ladder in the first and last lanes for an example). These are all 1-2% agarose in TAE, stained with ethidium bromide. I've never seen issues like this in my previously lab and I can't think of an obvious difference in the setup here. I'm running short gels (7 cm) at 70-90 V.
Has anyone else seen this issue and know how to avoid it?
Thanks.
it could be an effect of thermal diffusion broadening the smaller bands as the gel heats up so you could try running the gel in a cold room or just at a lower voltage and for a longer time
it could be an effect of thermal diffusion broadening the smaller bands as the gel heats up so you could try running the gel in a cold room or just at a lower voltage and for a longer time
Hi Michael,
Just a thought in addition to Elsa`s comments. The `letterbox shape` arises from the well size and is inflated by a `relatively` high voltage compared to the rate (factor in agarose%, DNA weight, voltage, etc) at which the samples are running through the gel. Your voltage seems standard in reference to the size of the wells but you might want to start at a lower voltage (minimum 35 - 50 volts). Using smaller wells might improve your result.
Sometime it can be problem of running buffer (TAE buffer). You can prepare new buffer and run on lower voltage. Check the voltage fluctuation also because that might be a problem also.
Thanks everyone for your tips so far. Just to clarify Elsa, in this example it looks limited to the ladder but I have seen it happen to my samples too. I'll let you know if any of these suggestions help.
Hi Michael
Even if you want to run in TAE buffer (because you need to recover the bands, etc), a test in TBE should help you clarify whether thermal diffusion (e.g., too high voltage) is a problem. Depending on the amount of available buffer in the tanks, most of the cations in the upper part of the gel can move to the lower side of the gel, thus making the intensity to grow up (and the heat too, in order to maintain the voltage).
You could also re-circulate your buffer.
I have had this problem when I have too much DNA in the lane for the conentration of the gel. I think it is due to the current can't pull all the DNA through the pores fast enough so some is getting ahead of the rest. You can try loading less per lane (eg. 500ng marker) or using a lower agarose concentration (0.8%). Also, I found Envirosafe dye (from Helixx) rather than EtBr works better in this respect and much less of a health risk. You can also try staining after running rather than in-gel.
Which loading dye you are using ?
What is the content of loading dye with reference to glycerol ?
Use varying concentration of glycerol in BPB dye and mix with new molecukar weight marker.
Might be you are having same lot of loading dye for mol wt marker but your sample are not bad as compared to the marker.
Change the marker...and also the loading dye....
It seems that the concentration agarose is not suitable or prepared agar is corrupt.
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