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I had to digest my insert containing plasmid and another vector into which my insert has to be ligated, each with BamHI and NotI.

Upon digestion, I ran the gel to check for results. The digestion seemed to be ok the first time with reasonably good bands. Since I needed more concentration I ran the gel with the same reaction setup (30microL) for the second time.

Well 1 - 100bp marker

Well 2 - Insert in a vector (820 ng/microL, 3microL)

Well 3- Plasmid vector into which the insert has to be ligated (1000 ng/microL, 2microL)

The marker bands were faint while there were absolutely no bands in the other wells. What could be the reason for failure during the second run? I've performed the second time digestion in a span of 40 min after the first one.

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    $\begingroup$ Usually there is no issue of mixing. One possible reason is degradation. But you say marker is faint (marker wont get degraded). Stain the gel with extra EtBr and see again. Was it the same gel?? (If so then definitely stain it again beccause EtBr migrates towards the cathode) $\endgroup$
    – WYSIWYG
    Commented Jul 30, 2014 at 11:53
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    $\begingroup$ Can you show the gel image? Otherwise I support WYSIWIGs idea. There might not be enough EtBr left in the gel. What bands of the marker are visible? All, or only the highest? Additionally: Are there marker bands visible in the same size region as you digested DNA? If so, your problem is on the digest side. You are loading a ton of DNA on the gel, this should be visible. $\endgroup$
    – Chris ♦
    Commented Jul 30, 2014 at 12:08
  • $\begingroup$ Chris and WYSIWYG are right sounds like it degraded or was a bad batch of gel. I feel for you such are the things of nightmares. $\endgroup$
    – user1357
    Commented Jul 30, 2014 at 21:16
  • $\begingroup$ Ethidium will also break down slowly under fluorescent lights. It's usually not a problem, but I did an internship at a startup so poor that they reused the same gel over and over, so we kept it covered in aluminum foil and had to restain it all the time. But it looks like you're using enough DNA to see. Make sure the EtBr and gel and buffers are fresh. $\endgroup$
    – user137
    Commented Jul 30, 2014 at 21:21
  • $\begingroup$ @user137..EtBr doesn't get degraded soon.. It diffuses/moves out of the gel. $\endgroup$
    – WYSIWYG
    Commented Jul 31, 2014 at 5:07

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Usually there is no issue of mixing. One possible reason is degradation. But you say marker is faint (marker wont get degraded). Stain the gel with extra EtBr and see again. If you reused the gel then definitely stain it again because EtBr migrates towards the cathode.

It doesn't seem like degradation to me but to be cautious for the next time:

  • Use freshly autoclaved water. If you are not sure then filter using syringe and keep some 10ml for yourself
  • Ensure that the plasticware are also autoclaved properly.
  • Use endo - plasmid extraction kits, if available
  • Dissolve the plasmid in TE (only if conc is high and you need not put much in the reaction mix else EDTA interferes in the reactions. In that case use Tris-Cl pH-7.6 ).
  • If you have to digest a lot of DNA then set up small reactions in many tubes.
  • Do not incubate the reaction for a very long time (>6h).
  • Inactivate the enzyme by heating if you are not running the gel immediately.
  • Ensure that the gel running buffers are not too old.
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  • $\begingroup$ Agreed, seems like overall signal is week, so a stain/destain seems in order $\endgroup$
    – Atl LED
    Commented Jul 31, 2014 at 20:29

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