Hello,
I made an electrophoresis gel with the same plasmid (5828kb) but with:
- 2 different concentrations:
-in well 2-5; 0.5µg of DNA
-in well 3-4-6-7; 1µg of DNA
- 2 different manufactured batch of plasmids (perform by 2 different suppliers):
-in well 2-3-4: first suppliers
-in well 5-6-7 the second suppliers
I have the nicked form (second band) and the supercoiled form (third band). But I don’t know what is the first band. This band more important with the first supplier batch. Can you help me to explain this band?
do have any explanation?
Moreover, my A260/A230 ratio is a little too high (2.3) but I have the same result in both suppliers. do have any explanation to explain it? What could be the impact on my electroporation efficiency?
Thank for your help
Dear Marine,
Identifying plasmid bands from uncut DNA samples on a gel is always a problem, and depending on the isolation protocol, age and storage of the sample, you can have more nicked (relaxed) and supercoiled forms, along with degradation smears. I find it better to check the quality of my plasmid preparations by linearising them with an appropriate restriction enzyme - or even cutting them in to a series of fragments - and then looking at the results on a gel of the appropriate gel concentration.If you know the restriction map of the plasmid, you have a clear idea of the bands you expect to see - and if they are nicely within your DNA ladder, you can determine sizes easily. Contaminating DNA will be obvious too (degrade DNA may disappear during the restriction incubation though).
Andrew
Hello Marine,
the top band could result from a plasmid dimer. To confirm this you could digest the plasmid with a restriction enzyme that cuts once in your plasmid and check that you get a single band migrating as expected for the size of your plasmid. As to the A260/A230 ratio, a low ratio would suggest contamination with guanidine thiocyanate, but in your case, if you didn't flip the ratio (i.e. A230/A260 instead of A260/A230), a higher ratio shouldn't affect transformation efficiency. At any rate, if it is E. coli that you want to electroporate, the amount of DNA that you need is so small that even a significant contamination should have no impact on transformation efficiency
The second band does not match 5828bp, but appears to be about 7500bp. I have no idea what this is.
The second band does not match 5828bp, but appears to be about 7500bp. (I guessed that 5828kb is a misspelling of 5828bp.
I can't identify the band around 5800bp.
The third band is designated as a supercoiled, which I think is the nick form of the obtained plasmid in these suppliers. I would also guess that the very thin band further down is the supercoiled form (fourth band).
Unfortunately, therefore, I'm sorry to say that you got the plasmid you were expecting.
I suspect that what is shown as a strange band is the bacterial DNA fragment that is seen when a lot of E. coli genomic DNA is mixed in during plasmid purification. If the alkaline prep method is used as the plasmid purification method, the alkaline treatment time should be strictly 5 minutes to improve the results.
As Andrew and pierregaga mentioned, I think it would be a good idea to evaluate cleavage by restriction enzymes, showing plasmid maps, etc.
Best,
Narumi
Well, it looks like you have lots of plasmid. Try a few restriction digests to see if it fragments in the expected pattern. Cutting 1 time should clear up any nicked/supercoiled DNA and let you see if that really big band was your plasmid or not. Any chance it's bacterial genome?
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