I have subjected the same DNA with single digestion and double digestion using different buffers to compare the efficiency of the buffers. I have loaded the uncut DNA (maxiprep DNA containing, supercoiled and nicked circular forms of DNA) for comparison. But the single digestion (linear DNA) band although is above the double digested DNA (with the successful release of insert) doesn't match with the linear DNA band in the uncut sample. Rather, it appears along the supercoiled band. Does it mean that the single digestions have not worked?
Picture is given for reference.
lane 1: 1Kb DNA ladder
lane 2: uncut plasmid DNA
lane 3, 4: single digestion with enzyme 1
lane 4, 5, 6: double digestion with enzyme 1 and 2
Hi there,
The digests are fine. The downshift you observe for the linearized plasmid when double digested is due to the liberation of the insert.
Thanks Dominique. I understood that, my question is that, the linearized DNA in single digests align with the uncut DNA's lowermost band, which I assume to be supercoiled plasmind. The upper band could be the linear DNA there. So, Does it mean that, the uncut DNA is already linearized and there is no supercoiled DNA in it?
Hi,
It looks as if your digestion worked well. Technically, the success of the double digest indicates that your single digestion was successful. The intensity of your uncut plasmid however can make it difficult to accurately judge the real band size as there seems to be a smear in the band. If I were you, I would run the gel with the same concentration of uncutted and the digested plasmids.
Goodluck.
Hi again,
From the single digest you get a single band running a bit heavier than the supercoiled form of the uncut plasmid (if you want to get sure about this, load exactly the same amount of uncut and cut plasmid onto gel and run for longer time)... So it's fine!
Hi,
The photo of plasmid digest gel you posted here showed all the correct digest pattern of an uncut DNA and its single and double digestion products . Uncut DNA (supercoiled DNA) band is always a bit downshift compared to the single cut band of supercoiled DNA,even though you subject the same amount for both, it is fine as long as the level of single band is in the same level of top edge of uncut band.
and I agreed with one of above comment that's that your double digestion products confirmed the success of both single and double digestion in which the top band of double digestion should be a bid lower than the single digestion band ( linearized plasmid) because the liberation of the insert from the plasmid, you can valid this via looking at this plasmid map which the sizes of the top and lower band of double digestion( backbone and insert) should be the same as the size of whole plasmid( linearized DNA in single digestion).
hope it may help!
I agree with Yifu thetop band of double digestion should be lower than the digestion band.
hi there,
i agree with Yifu about the top edge of the band tallying with the edge of the uncut plasmid DNA ok...so the single digestions are fine
As for the question of linearisation, how do you know that the bands in the uncut plasmid are of supercoiled and nicked circular. I guess the lowermost is that of linear plasmid because I dont find all the forms of plasmid isolated here as there are only 2 bands in the uncut lane
also for future one must load the same amount of cut and uncut DNA when carrying out linear digestions
take care
Dear Tanushree,
Can you conclude from the uncut plasmid's mobility on gel that there isn't any supercoiled form present? Is it necessary in all DNA preparation to find all the three forms of DNA? Because, in my DNA preparations so far, linearised DNA is absent many a times. Experiments with that DNA have worked successfully! Can't you say anything about you till you see all three bands!
Hi,
Yes thats exactly what I was trying to say!! You assumed that the lowermost band in your uncut DNA is that of supercoiled. I would say its not because ALL FORMS OF DNA DO NOT GET ISOLATED necessarily during plasmid preparation. Since your single digestions give a single band high chance that the band in the uncut plasmid is that of linear DNA itself tallying with your single digestion!!....you have run a ladder besides..so to see whether the DNA is fine or not match the size of the single digested product with the ladder....if thats the size of the DNA its perfectly fine!!
DNA preparations vary each time you prepare DNA, so do the forms of DNA that are isolated. High chance that this DNA preparation has a linear band!!
From the gel of the uncut plasmid and from the digested products its apparent that there's no supercoiled form present here!
Tk cr
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