I had done an amplification of pet28a plasmid to introduce some mutations in it using the designed primers.
Pcr product was gel eluted, then sequentially was treated with Dpn1 and PNK enzymes. This was then transformed into DH5 alpha competent cells and plated. I have got colonies in these plates and had isolated the plasmid. But, it's a smear on the gel. It's concentration on nanodrop was ~850ng/ul
In the picture added, 2nd well has plasmid of concentration 850ng/ul and 4th well has 200ng/ul plasmid.
It would help to have a dna ladder on the gel but this looks more like RNA than plasmid DNA
It looks like there is no plasmid DNA present in the gel. Only degraded genomic DNA/RNA. Have you done the color selection as well as the antibiotic selection of the colonies? In addition, the procedure you have described is not clear.
I'm not seeing anything that looks like a plasmid. Did you try including a sample of just the pet28a as a reference?
A smear appearance of plasmid DNA on an agarose gel is often due to the presence of degraded or partially degraded DNA. This can occur due to a number of factors, such as exposure to heat, mechanical shearing during handling or pipetting, or exposure to nucleases.
In your case, the smear appearance could be due to partial degradation of the plasmid during the extraction process, or due to incomplete removal of impurities during the purification process. It's also possible that the plasmid has become nicked or partially degraded during storage.
To confirm the integrity of the plasmid DNA, you can perform a restriction digest and run the resulting fragments on an agarose gel. This will allow you to confirm the presence and size of the expected plasmid DNA fragment, as well as any additional bands resulting from partial degradation or incomplete digestion. You can also perform a gel extraction step to isolate the intact plasmid DNA from the gel for downstream applications.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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