4 replies to this topic

[inov]

Hi

This is my first post - I apologise if this may have been covered elsewhere.

My problem has been longstanding - occasionally I get smudged bands which are uneven on my 0.7% agarose gel.

I am cloning the DARC gene, and have amplified it after a maxiprep (qiagen) with good yields. Then I cut with pvu1, xho1 and xha1 overnight. I purify this with fermentas kit, run a gel and excise the gene which has been separated from plasmid. Then I gel purify (fermentas) and measure DNA concentration, finally followed by sequencing and a ecor1 cut to confirm the correct product.

Recently and suddenly all my bands have become untidy. There is some smearing in the lanes, some uneveness of the bands, and this was despite weekly changes of buffer. It happened between two of my maxiprep digests which I was excising the gel from. My and my collegues' gels were unitdy. The ladder and the uncut product is similarly affected which makes me think it could be:
1. some buffer problem but was recently made up (2 days ago)
2. something in the water used to dilute but was a new water opened last friday
3. some contamination in the agarose melting flask which goes into microwave - this has been washed today.

Is there anything else I can try to fix this up? It has happened intermittently before - goes and re-occurs.

Thanks for anyone's help /advice

Inov

[phage434]

Make sure the gel buffer and tank buffer are the same. Make sure you are using buffer to make your gels(!). Excess heat from running at too high a voltage can cause this problem. Check the platinum wires in the gel tank. A broken wire can make the gel run unevenly. Is your gel box level? Is the gel poured on a level surface?

[inov]

phage434, on 15 December 2010 - 09:07 PM, said:

Make sure the gel buffer and tank buffer are the same. Make sure you are using buffer to make your gels(!). Excess heat from running at too high a voltage can cause this problem. Check the platinum wires in the gel tank. A broken wire can make the gel run unevenly. Is your gel box level? Is the gel poured on a level surface?

Thank you phage, my voltage is 80, and the buffer is all the same stuff (0.5% TBE). I will check that the gel box is level. Wires seem ok, but I will get someone technical to have a look. I've attached two pictures so that you might see what I mean.

Thanks again

inov

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[ElHo]

Two ideas:

1. Removing the comb too early, leading to damaged slots.
2. Two much salt in your DNA samples.

[inov]

ElHo, on 16 December 2010 - 08:27 AM, said:

Two ideas:

1. Removing the comb too early, leading to damaged slots.
2. Two much salt in your DNA samples.

This is great - it seems to have worked - leaving the comb in for a bit longer.

I haven't done anything in the salt - how would I do that for future reference?

Thanks

Inov