I normally inoculate 5 - 7 colonies from selective antibiotic LB plate after transformation in the liquid LB with the same concentration of the same antibiotic. The next day, I do plasmid miniprep for plasmid isolation and then run a restriction digest for all of them and go for the pattern that is expected (from VECTOR NTI). If found, I sequence and confirm it, then save it in the stock with the proper numbering and labeling.
Later, I pipette 1 ml of the culture from the above liquid LB media of the same plasmid that has shown me the expected banding pattern and inoculate again in 130ml of LB and the same antibiotic (and the same concentration). I do plasmid midiprep and isolate a high concentration of the Plasmid.
Out of curiosity, I run the undigested and restriction digested miniprep and midiprep to confirm. Here my undigested shows the similar results but my redigestion of midiprep varies from the miniprep and the expectation. Any comments on this?
There are a few possibilities. It is certainly possible that some sort of deletion even occurred when you grew the midi-prep and that explains the difference. It might also be that you accidentally inoculated from the wrong tube. However the most likely answer is that one of your DNA preps is slightly contaminated with something that is inhibiting the restriction enzyme, or has too much salt in it. So what you are seeing is an incomplete or partial digest from one of them. Without seeing a picture it is hard to tell.
Have you considered partial digestion? Your midiprep will be more concentrated than miniprep and if you did not consider the concentration while setting up digestion, you might get lot of partials.
How does the midiprep vary in the digestion pattern? Are you seeing larger fragments than you expect or smaller? Have you repeated the midiprep and gotten the same results just to make sure it wasn't a fluke? If you could post a picture that might help in the diagnosis. From what you have said so far I think the partial digestion is a strong possibility.
HEY ! HERE IS THE PICTURE OF THE DIGEST
The undigested looks like Super-coiled !
You have three different types of DNA here. Nicked circles, linearized DNA, and supercoiled. You only have a partial digest. I don't know what concentration you are digesting using the 0.75 uL of enzyme but I would decrease the amount of DNA. In a 50 uL reaction volume I usually use 1 uL of enzyme, you might try increasing to that.
It looks like a really poor digest, are you doing it in the right buffer and at the right temperature? I agree with kiev thatyou could try to digest less. Another thing that helps restriction is adding more enzyme after an hour or so and digest it for another hour, since it does lose activity the first bit may not be enough to digest it completely but still make it more accessible for the second batch (especially for difficult genomic DNA). Are you expecting 450, 550, 600, 1100 and >7000 bp?
Hey wht Henk-jan is telling I agree with it...you check the buffer you are using....How much enzyme u r using.......whts time period? You can increase quantity and incubation time...also go for second time digestion with using same batch plasmid and newly extracted plasmid....and check difference
y u r getting one more band above 5000bp...Is it genomic DNA contamination? which kit you are using for isolation of plasmid?
Kiev and Henk-Jan are correct. You have a partial digest of the midiprep, and probably because you put in too much DNA for the amount of enzyme. I routinely use 2U enzyme for up to 1 µg DNA, and digest for 2h. If the same reaction conditions (i.e., buffer and temperature) were used for all three digests shown, then it is likely to be the amount of DNA, and possibly something from the kit you used to midiprep. I have found that DNA prepared using some kits is more difficult to digest. I am afraid that I don't know why this happens, but have assumed it is due to some component of the proprietary solutions.
I have to agree with the others, it looks like there is just too much DNA to get a complete digest from your midiprep. If I read your numbers correctly you are using about 2.5 ug DNA. I would reduct that to about 0.5 ug and see if that works better. You could also do an ethanol precipitation of your midiprep to remove any contaminants if you think that might still be an issue.
Your DNA is correct ! It has not been digested properly. In fact, if you have the same gel, increase the exposure time and capture the image; you will see the desired bands. I could see it by adjusting the contrast of the image. There is still lot of supercoiled DNA left in your gel. You can dilute the DNA and re-digest it and you will see the desired product.
For your reference, I have attached the edited gel picture. Hope this helps.
@Vikas, I thought I saw a faint smudge at the desired size as well, good idea to enhance the exposure/contrast. It may also help to load only half the amount of marker.
If you use less in the digest, still try to load enough to see the small bands
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