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Undefined bands in Gel


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#1 sdem11

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Posted 04 June 2013 - 11:53 AM

Hi, I've been trying to run gels for a series of PCRs to test primers. However the bands so far have been curvy, slanted and very unclear. I then tested a 100bp and 1kb marker in a gel I made to check that the error is from the gel electrophoresis and not the PCR. A similar problem occured however, and I can't seem to figure out what is wrong.

I'll attach both pictures. The first gel was run at 80V and is a 3% gel. The second gel is also 3% and it was run at 90V.

If anyone has any ideas on what to fix that would be great.

Thanks!

Attached Thumbnails

  • Gel 1.jpg
  • Gel 2.jpg


#2 hobglobin

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Posted 04 June 2013 - 12:02 PM

There are several possibilities: gel not completely set and used too early (I'd let it at least 30 min cooling down and set), agarose not completely molten (both if it's agarose), too old buffer, to mention very common problems.
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#3 sdem11

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Posted 04 June 2013 - 12:15 PM

I let the gel set for about an hour and then kept it in the loading buffer for an hour before loading. When making the agarose I heated it in the microwave for a minuted swirled it and left it for another 20 sec and it looked molten. I haven't changed the buffer in a week, however I did a gel the day I changed it and it was still very smeared. I've tried changing little things like the size of the well comb and have varied the voltage from 80V to 100V but nothing seems to make the bands defined.

#4 hobglobin

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Posted 04 June 2013 - 12:29 PM

You should look carefully if it's really molten and not any almost transparent particles or cloud-like stuff is left after microwaving (if you didn't do). And you made the gel with the same buffer as it's in the gel chamber (and didn't use water)? Did you try another gel chamber (perhaps uneven electric field)?
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#5 sdem11

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Posted 04 June 2013 - 12:47 PM

I want to say it was molten but I will double check next time to make sure it really is molten. And yes I made the gel using the same buffer that I poured into the gel chamber and didn't use any water. I have tried another gel chamber once but did not see a change in results however I suppose I could use the different chamber than the previous two and try again. If you have anymore suggestions please let me know.

Thank you!

#6 phage434

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Posted 04 June 2013 - 12:48 PM

3% agarose is very high concentration. The most likely thing has already been mentioned, which is failing to completely dissolve the agarose. Make absolutely certain that the solution is crystal clear and uniform before pouring the gel. A 1.5% gel would work better for 100 bp and 1000 bp fragments.

#7 sdem11

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Posted 04 June 2013 - 01:43 PM

Here is another example of one of the gels I did. It was run at 100V using a 2% gel so I don't think the fact that the others are 3% is what is causing these types of bands. Maybe this picture could help to understand what is going wrong, but thanks I'll definitely be sure to check the agarose is completely molten.

Attached Thumbnails

  • Gel 3.jpg


#8 bob1

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Posted 04 June 2013 - 02:08 PM

Some of that wiggle in the bands is almost certainly from the wells being torn when you removed the combs. Make sure the combs are clean before you pour the gel and remove carefully by pulling upwards, lateral movement can tear the wells.

#9 mdfenko

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Posted 05 June 2013 - 04:37 AM

also, make sure there are no bubbles in the gel or well or sample (after loading). don't puncture the surface of the well with the pipette tip.

one other thing, it looks like the gel is slightly off the axis of the apparatus (the lanes are running at an angle). make sure that your gel is perpendicular to the electrodes.
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#10 leelee

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Posted 06 June 2013 - 12:24 AM

Looks to me like you have stuck your pipette tip down into the well, damaging them as mdfenko has suggested.

Don't do this- hold it just above and allow the dna+loading buffer to sink into it.

For melting and/or dissolving my agarose, I heat until starting to boil, then swirl, then heat again a little further if I can still see fibres in the molten mix when I hold it up to the light. I then let it cool until I can hold it without burning my hand, and swirl again really well before pouring.




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