Dear Viewers,
I purchased the commercial pET28a plasmid, and when I ran it on an agarose gel (200 ng), it showed three bands, which is expected due to its shape. When I performed a single restriction digest (R&D) with EcoRI, it produced only one band, confirming that I have a pure pET28a plasmid.
Next, I transformed this pET28a from the commercial stock into DH5α cells, harvested the plasmid, and ran it on a gel, which again showed a single band. However, when I did a single restriction digest of the harvested plasmid with EcoRI, there was no band present.
The potential issues I considered were:
1. Low concentration of the plasmid,
2. EcoRI may not be functioning,
3. Possible issues with the buffer,
4. Problems with the gel.
I increased the concentration of the plasmid, but I still did not observe any bands. I tested options 2, 3, and 4 on the control, and I did get a band in the control.
Can anyone help me come up with a solution?
How did the rest of your gel look? Nice bright ladder? If that all looked good then you can at least rule out omitting gel stain (I've done that) or the UV light burning out (yep, had that happen too).
My first suggestion would be just to try it again. Run your gel with the original plasmid (if you have any left), the isolated plasmid, and the cut isolated plasmid. It's an easy double check.
Dear Amy Klocko Thank you for your kind reply.
Yes, I received the pure and bright band without treatment with Restriction Enzyme. If I have the pure plasmid, then why should I isolate it from the gel?
And my question was, why is it happening even after trying the above-mentioned troubleshooting?
Hmm, that is a bit of a puzzle. So, to sum up. You were able to transform the plasmid into cells and if you run this plasmid on a gel it gives a nice band. But, if you digest this plasmid with EcoRI the band no longer shows up. Ok, sounds like a digestion issue.
My guess is somehow one of the digestion reagents (enzyme or buffer) got cross contaminated with something (like another enzyme) that is now chopping your plasmid into tiny fragments.
Try the digestion again but with a fresh source of buffer and enzyme, perhaps borrow a bit from another lab or order it fresh.
Dear Amy Klocko,
I have tried using different enzymes and buffers, but the issue remains unresolved. Additionally, the enzyme and buffer are not cross-contaminated, as they work correctly with the control group (another plasmid).-contaminated because they work fine with the control group (another plasmid).
Hmm, very puzzling. Do you need to use EcoRI in particular for cloning with this plasmid?
What happens if you digest the plasmid with a different enzyme?
A single EcoRI digest of pET-28a linearizes the plasmid, producing one ~5.4 kb band on an agarose gel. Undigested plasmid shows supercoiled and open circular forms. Incomplete digestion or contamination may cause additional bands. Ensure proper digestion conditions and verify enzyme activity to avoid anomalies. For double digests, multiple bands appear, but single digests yield only one linearized fragment.
@amy klocko
Yes I have to use this particular restriction enzyme because my gene of intrest is contained with this enzyme (restrictions site) so for stick ligation I particularly need this enzyme digestion
Hmm, if you are needing EcoRI then I would suggest a re-try but with a fresh batch of enzyme and buffer.
Can you put a gel picture to show the uncut and cut DNA so I can see what you are seeing?
As a note, do you run an uncut control? One where you set up everything as if it were a digest but without the enzyme? You incubate it with your actual digest too.
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