Hello Everyone
I am trying to isolate pET28a plasmid (5.37 kbp) from E.coli strain. The electrophoresis result shows only one band that is >20 kbp. This is triplicated and same result comes all the time. I took positive control of pGLO strain and found expected band of it, which implies my protocol is fine. My pET28a strain was grown in kanamycin added media (solid and liquid). INTERESTINGLY, when I digested that >20 kbp band with enzyme (single cut) it showed single band of approximate disired size.
Do you have any idea regarding my error or anything else?
First of all, the only thing that really counts is the size of the band after digestion. If you run uncut plasmid you can get a variety of bands, supercoil, nicked circle, dimers, trimers etc. So it is hard to predict much. I agree that 20kb sounds unusual but if you digest and you get the right size band (and only that band) it probably is ok.
I would however add that your gel showing the digested band is not terribly convincing, you have so little DNA in the lane that you might be missing other bands for example.
Lastly, you obviously have a whole lot of material running low in the gel. This is normally RNA which should be removed by RNase but it might also be degraded DNA. I would first check to see if your RNase solution is working because it really should go away with RNAse treatment. The other point is that having so much materials in the lane, it may have an effect on the observed mobility of your bands.
So in the absence of a clean gel with sufficient DNA, I'm not sure I would jump to any conclusions.
Hello, the single weak band can be the result of primer dimer formation during the PCR reaction.
It is possible to get only multiple primer bands in a gel if the primers are annealing at multiple sites on the DNA template. This can result in multiple bands appearing in the gel, rather than a single specific band. However, based on your description, it seems that you are not seeing the expected band for your pET28a plasmid.
One possibility could be that the plasmid is not being efficiently extracted from the E. coli cells. You could try optimizing your DNA extraction protocol to improve yield and purity of the plasmid. Another possibility could be that the plasmid has degraded or has mutations that prevent it from being amplified.
It's also worth considering if there may be a problem with your PCR reaction, such as suboptimal annealing conditions or an incorrect ratio of primers. You could try adjusting your PCR conditions or using different primers to see if this improves the specificity of the reaction.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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