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?
Dear Sanjeev Kumar,
What did you observe on gel when you loaded EcoRI restriction of harvested plasmid? Was it 3 bands, or no band (nothing) at all?
Dear Masooma Hammad,
As I mentioned in the description, I did not observe any bands after treating the plasmid with the restriction enzyme.
But it's obvious, without treating the plasmid, it is expected that we can see three bands based on their shapes, as I also noted in the description.
Dear Sanjeev Kumar ,
If you got plasmid on gel after transformation but you got NOTHING after its restriction and as you have mentioned that you have already sorted all the issues related to plasmid concentration, enzyme, buffer, and gel. I would suggest.
1. Use another enzyme(s) for confirmation
2. Do another transformation with some other stock of cells
3. More importantly, recheck the type of your competent cells. Were they actually DH5α cells and not BL21 type cells (or other of similar type)? Because such issues arise if you harvest plasmid from such cells, which have nucleases in them. These nucleases come with plasmid during extraction especially if you are doing miniprep with solutions (not kit). and when you do restriction reaction, these nucleases get a favorable environment (buffer) for their action and cleave your plasmid, which will not appear on gel after restriction.
Good luck with your work!
Dear Masooma Hammad thank you for the favourable suggestions. But I already performed these experiments (which you suggested). And cross-confirmed that everything is working fine with the control group.
How much plasmid did you use for the digest and how much did you load into the gel?
You need a minimum of about 10 nanograms to just barely be able to see DNA on an agarose gel with ethidium bromide, and that's with a good light source in complete darkness.
My guess is that between digesting (possible cleanup?) and loading, you basically diluted the DNA so low you can't see it.
Dear @katie Brunette
I loaded about 500ng and more importantly I didn't cleanup the digested product, I directly loaded on to gel so there was no chance to possible cause of cleanup.
- Badges
- Science topic
- Similar topics
- Filing