I ran 0.7% agarose gel electrophoresis of my plasmid DNA sample consisting of GFP vector, using NheI and HindIII restriction enzyme.
I used two types of plasmids, i.e., isolated by miniprep-alkali method, and another isolated by midi prep system (pure) of the same source E. coli.
The 4 and 5 lines show cut and uncut plasmid, respectively, isolated by midi prep system.
The 1, 2, and 3 lines show the 500 bp marker, cut, and uncut of miniprep isolated plasmid signal, respectively.
In the attached image, we found an extra band in the plasmids isolated by alkali method (line 2 and 3), but in the pure samples (line 4 and 5) no such extra bands were found.
What are the possible reasons behind the result? How may I interpret this result? If anyone has experienced similar results, please share.
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$\begingroup$ Not sure what it is, but I noticed this today as well on a gel. It was only present in plasmids (digested) that were grown in DH5alpha (and absent in the XL1 blue). Could be coincidence though. $\endgroup$– Geo VoglerJan 6, 2017 at 23:26
2 Answers
Looks like genomic DNA contamination to me.
More importantly, does it really matter? I certainly appreciate the curiosity but, if your objective is cloning, I would just keep going and then do some diagnostic cuts of your plasmid after transformation. You can clearly see your vector or insert in the gel.
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$\begingroup$ @SanjuktaGhosh Than answering as comment, maybe it is better to write directly the answer? As well as it doesn't look like a short hint but a really small answer because it seems there is not much things left to elaborate or cite. $\endgroup$ Feb 8, 2017 at 20:37
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$\begingroup$ If it was gDNA contamination I would expect to see many more bands... $\endgroup$ Feb 8, 2017 at 20:42
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$\begingroup$ @JoeHealey I would too but, to me, it's the most plausible explanation for such a large fragment. $\endgroup$ Feb 8, 2017 at 22:19
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$\begingroup$ Especially given that it is absent in the so-called "pure" fractions, which are presumably cleaner. $\endgroup$ Feb 8, 2017 at 22:32
Could be several things...just off the top of my head. Without your vector map and expected sizes it will be almost impossible to say for sure.
You'll probably also have to give us more information about your incubation conditions etc.
- Your original culture you prepped from might not be as homogenous as you think:
- You've ended up with a mutant (perhaps in your restriction site in that particular clone)
- Your original culture is contaminated, perhaps with a second E coli bearing a different plasmid.
This is probably unlikely, assuming your molecular/microbiology technique is decent, but does happen from time to time.
Incomplete digestion. Something in the eluate of your first extraction method has possibly inhibited your enzymes, that isn't happening in the other extraction mechanism, or you didn't leave the digest for long enough.
You missed something out of the reaction mix for one of the samples (I usually blame myself first when things go wrong so I usually just repeat it to be sure!)
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$\begingroup$ How an incomplete digestion give rise to a molecular size far bigger than entire plasmid (track 4 and 5)? $\endgroup$ Feb 8, 2017 at 20:40
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1$\begingroup$ Uncut plasmid can run at all sorts of lengths. If it's supercoiled, it will run further than it 'should' for its size. If it's circular but not supercoiled, it will run slower than it 'should' vs if it was a single linear fragment as it doesn't move easily through the gel. $\endgroup$ Feb 8, 2017 at 20:45