I tried transforming my Ligated gene into the competent cells but nothing grew at all. So when we finally had some colonies, we did plasmid miniprep to increase the concentration. We need more miniprep of our ligated gene. So, we tried to transform our minipreped gene so that we can get colonies. We did get colonies, we did plasmid miniprep for the colonies and tried to verify but we see more bands than what we should be doing. What are we doing wrong?
Hi Ramyasri,
I am a little bit confused by your description. Once you obtained colonies after transforming the ligation mixture, you isolated the DNA from single colonies and then transformed each individually into a fresh batch of competent cells?
If your plasmid has not been digested with restriction enzymes then it will run according to its supercoiling 'status'. In a typical DNA prep the fastest band corresponds to the supercoiled plasmid, and the slowest one to the relaxed plasmid (which is presumably nicked).
Unusual shifts from restriction digests may occur if the enzyme stays bound to the DNA.
However, if your restriction digest clearly points to the fact that you have multiple plasmids in you mixture, then it is likely that the colony you picked for DNA isolation actually contained multiple clones, which were not apparent from the shape of the colony. This can be easily solved by re-streaking this colony and isolating the DNA again from clearly separated colonies.
Hi Ramyasri,
Did you send the plasmid that you made/obtained in for sequencing? Just because you performed a ligaton reaction on your vector and gene of interest, does not automatically mean that the gene was inserted properly.
Did you also doublecheck the restriction sites? It has happened to me that I used a vector that also contained the used restriction sites in multiple locations other than the intended one.
There also might be some aspecific restriction which may explain some very light bands. This could be influenced by the buffer that you used.
Another explanation for different band heights after separation is, as Ana Crnkovic mentions, supercoiling. I've found that performing an enzyme digestion for about 4-6 hours minimizes the aspecific bands due to this issue.
Hopefully this helps!
supercoiling makes the plasmid run different. If you linearise them, they may all show you a correct band.
It could be the isoforms of the compaction of the plasmid, or maybe your ligation went wrong if you are using restriction enzymes before, your plasmid maybe have different restriction sites that you didn't know or the enzymes are having some star activity. Other usual problem is when you or your lab partners accidentally mix the enzymes or plasmids, or when you let the enzyme work for many hours.
I hope this response help, good luck.
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