I have used DH5a cell to increase the copy number of my plasmid. I have used a commercial kit for plasmid purification. However, after doing agarose gel electrophoresis, I get strong bands of fragmented DNA and a weak band of plasmid. What might be the probable solution?
Your gel shows undigested plasmid at the top and RNA at the bottom. It is not fragmented DNA.
Just do a digest to linearise your plasmid and use loading dye with RNAse to run on the gel again and I'm sure you get a sharp band at the expected size.
If the DNA is degraded, you will see the sharp band and an additional smear emanating downwards from the band, being the most intense close to the band and getting progressively weaker towards the bottom.
Your kit is not very good, there should not be RNA in the eluate. We never see RNA when we use a commercial kit (i.e. Wizard prep).
Your gel shows undigested plasmid at the top and RNA at the bottom. It is not fragmented DNA.
Just do a digest to linearise your plasmid and use loading dye with RNAse to run on the gel again and I'm sure you get a sharp band at the expected size.
If the DNA is degraded, you will see the sharp band and an additional smear emanating downwards from the band, being the most intense close to the band and getting progressively weaker towards the bottom.
Your kit is not very good, there should not be RNA in the eluate. We never see RNA when we use a commercial kit (i.e. Wizard prep).
Thank you for your answer. Actually, I want to isolate whole plasmid (not a digested one)
Hi, Jurgen explained you perferctly what happened.
I attached an example of agarose analysis extracted from qiagen plasmid purification handbook that can clarified you what "went wrong" during plasmid purification. It helped me a lot in the past!!!
Hi Mohammad, you isolated whole plasmid with your kit but to control your plasmid extraction you have to digest it and run on agarose gel in its linear form. In this way you can control your plasmid integrity, contamination from RNA (as in you case) or genomic DNA from DH5.As Jurgen said, your kit can not good, expired or you have problems during some part of the protocol, so you have always to check your plasmid after digestion.
Yes Mohammad, you isolate a whole plasmid, and you use only a very small portion of your preparation (for instance 1 microlitre out of a total of 100) in quality control. Then you have 99 microlitres of whole plasmid left to carry on with what you wanted to do.
I usually use an enzyme that linearises the plasmid. I never run undigested plasmid, supercoil and multimers don't stain well and don't make sharp bands. Linear DNA gives a sharper, brighter band, and when you see no smear, there is no degradation.
Jurgen, I wanted to send the whole plasmid for DNA sequencing.
Why there is RNA band ! ! !
Commercial kit contains RNaseA.
Did you add RNase into the solution before use?
To isolate band of interest (separate band out from other unwanted bands) for seq. we may use Wizard® SV Gel and PCR Clean-Up System (Promega) to (i) excise band of interest, (ii) purify and (iii) elude DNA.
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