Enzyme digestion resulted in extra bands from plasmid miniprep product - (May/16/2014 )
I was expecting 4 clear bands from my enzyme digest but saw almost twice as many bands from what is expected. Can anyone explain what might have caused this and how can I improve that?
Just a side note, I also ran the undigested plasmid in the same gel and there was a single band at the right size.
My enzymatic reaction was: 2h at 37°C (this was suggested by the person I got the plasmid from) with 2 units of BamH1 in a 10ul reaction with 1ug plasmid. The final mix contains 1x BSA and 1x buffer.
Can you state size of linear undigested plasmid, linear plasmid, sizes of expected bands and sizes of observed bands? Did you used FastDigest or normal RE?
Inbox on Fri May 16 08:57:35 2014 said:
Can you state size of linear undigested plasmid, linear plasmid, sizes of expected bands and sizes of observed bands? Did you used FastDigest or normal RE?
From the top of my head, linear/undigested plasmid = 16kB, size of expected bands = 9kB, 8kB, 7kB and 4kB. But instead I got many bands ranging between 9kB down to 2kB. Possibly 7-10 bands of various intensities.
Another question is do the restriction products always add up to the linear plasmid size? Seems like the 'predicted' cut products given to me by someone else doesn't add up to the expected 16kB (see above).
Partial digestion can give hard to interpret banding, but if some of your fragments are below 4 Kb (size of your smallest expected fragment) then something else is happening. Contaminants in the restriction digest (usually from the DNA prep) can cause star activity. You should do the digestion with the DNA fragment composing a small fraction of the total volume (also, the enzyme). If this does not change the result, I would strongly recommend sequencing your plasmid. Most likely the sequence is not what you (and others) think it is.
phage434 on Fri May 16 12:50:49 2014 said:
Partial digestion can give hard to interpret banding, but if some of your fragments are below 4 Kb (size of your smallest expected fragment) then something else is happening. Contaminants in the restriction digest (usually from the DNA prep) can cause star activity. You should do the digestion with the DNA fragment composing a small fraction of the total volume (also, the enzyme). If this does not change the result, I would strongly recommend sequencing your plasmid. Most likely the sequence is not what you (and others) think it is.
Could you please elaborate on "do the digestion with the DNA fragment composing a small fraction of the total volume"?
Did you mean to take the resulting fragments and do another digest?
No, I mean you should do an enzyme digest like this:
DNA: < 10 ul
RE buffer (10x): 5 ul
Enzyme(s): 1 ul each
Water: sufficient to bring the volume to 50 ul
As your DNA amount goes up, the chance for enzyme inhibitors becomes a real issue. Enzyme (really glycerol) concentration can also be a problem if people try to do low volume digestion.
phage434 on Fri May 16 13:17:59 2014 said:
No, I mean you should do an enzyme digest like this:
DNA: < 10 ul
RE buffer (10x): 5 ul
Enzyme(s): 1 ul each
Water: sufficient to bring the volume to 50 ul
As your DNA amount goes up, the chance for enzyme inhibitors becomes a real issue. Enzyme (really glycerol) concentration can also be a problem if people try to do low volume digestion.
The reaction I was using was:
DNA: 1ug (0.5ul)
RE buffer (10x): 1ul
Enzyme: 2 units (=0.1ul)
BSA (10x): 1ul
Water to make it up to 10ul
Do you think by doing a small 10ul digest, I would increase the chances of the non-specific fragments? I did just enough to load onto an agarose gel.
How much DNA (in ug) would you add to a 50 ul reaction?
Your reaction should work fine, but there might be too much DNA for your enzyme amount. This would not produce the bands you describe, so the problem is likely elsewhere (sequence).
The difficulty with low volume digestions is that it is quite difficult to pipet small volumes with any accuracy.
You need less than 20 ng in a band to visualize it. This usually means you need only 200-300 ng total in a digestion, even if looking for relatively small bands.
That DNA is quite concentrated. Where did it come from?
phage434 on Fri May 16 13:50:26 2014 said:
Your reaction should work fine, but there might be too much DNA for your enzyme amount. This would not produce the bands you describe, so the problem is likely elsewhere (sequence).
The difficulty with low volume digestions is that it is quite difficult to pipet small volumes with any accuracy.
You need less than 20 ng in a band to visualize it. This usually means you need only 200-300 ng total in a digestion, even if looking for relatively small bands.
That DNA is quite concentrated. Where did it come from?
Its a Miniprep product which I reconstituted to a concentrated solution with the idea that its easier to dilute it vs. having to concentrate it because it is too diluted.
When you concentrate DNA by evaporation, the contaminants are not removed, so concentrated DNA in this form adds contaminants. Better would be to ethanol precipitate and resuspend. Make certain you remove all of the ethanol. But your original miniprep likely could be added at 10 ul to a 50 ul reaction as above.