I was working on a gene construct synthesized in pet29 vector as a clone. Primers were prepared and optimized with gene at Tm 58 degrees. Once primers were optimized, I carried out transformation in expression vector and checked colony PCR with the same set of primers. After some time, I needed to conduct PCR for the same gene again for TA cloning and repeated BL21 transformation but issues occured. My primers that were previously optimized didn't work on the same gene on the mentioned Tm. After numerous trouble-shootings, I decided to check either the problem has appeared in my gene construct or not. I checked my commercially synthesized cloned gene on agarose gel in the intact and digested form and there was no band of gene once visualized. Is there any chance that my clone is destroyed by nucleases? What can be the reason for such conditions? It will be a great help if you can guide me
It could be that your "clone" was actually a mixture of some plasmid without insert and some plasmid with insert. At some point you selected one of the no plasmid colonies.
I would go back to your original stock of that clone, if DNA then retransform and if frozen culture then streak out for single colonies. And then prepare a new stock from a single colony/clone that you have confirmed by miniprep actually contains the correct plasmid with insert.
Ok so you digested the commercial vector with the gene in it. And ran it on a gel.
did you get a band showing the digestion worked? The undigested form is circular and will have multiple bands due to coiling. The digested form should have 2 bands, both different from the undigested, one showing the backbone and one showing the gene you cut out with the endonucleases. Or if you only used one enzyme, you will have one band in the digested lane.
If you used two nucleases and thus expect two bands after digestion, and got none, something went wrong with your loading of the gel or your digestion because there is no DNA. If you used two nucleases and got one band - and it is the expected size of the backbone minus the gene, but you have no gene band - this is unusual. It might mean that there was no insert in the backbone. So what you cut out with the nucleases is a very small segment of the multiple cloning site of an empty plasmid, and that segment has run all the way off the gel. OR it means that your plasmid was a mixture of empty backbone and the desired plasmid, the plasmid with the gene in it - and the proportion was something like 95% empty plasmid, 5% plasmid with gene, so the band for the gene is much more faint than the band for the backbone, and to see it you should use a GBOX and turn up the exposure.
Ok - when you first used this plasmid, you got it from a company, and do you just PCR the plasmid straight from the commercial tube to check the gene was there and then transfect cells and do colony PCR on those? and then just assume the plasmid was pure? or did you then do mini prep with a positive colony, and sequence the resultant plasmid, to make sure you have a pure stock?
Because if you did not do the mini prep and have just been using the original plasmid shipped to you, it might be a mixture of empty backbone and desired plasmid.
im guessing you repeated the transformation the second time and did colony PCR and got nothing - might just be a bad transformation?
Did you then PCR the plasmid?
if you didn’t, do that. If you have a band, your transformation was bad.
if you did, which I assume you probably did, and got no band for your plasmid stock from the company, then what might’ve happened is
the plasmid you got was a mixture of empty backbone and plasmid with gene. When you did the first transformation, you got lucky and got a bunch of the plasmid with the gene in the portion of the stock you aliquoted for that.
now your stock has gone from 95% empty and 5% gene to 99% empty and 1% gene. So your chances of a good transformation are lower
Do your primers bind the backbone around the insert or do they bind the gene? If they bind the backbone, and you did this PCR on the plasmid stock and got no band, the empty vector is outcompeting the one with the gene. try turning up the exposure on the GBOX, you might have a faint gene band. Or it’s outcompeting it so much there is no band. You need primers that amplify only the gene to confirm some plasmids have it.
if the primers bind within the gene and you’re getting nothing, your plasmid stock is a bunch of empty vector.
I recommend, if it turns out the stock is a mixture, you do a transformation and do mini prep to get a pure stock.
It will be ok. Best of luck. Cloning can be confusing. You’re going to figure it out and then you’ll be able to help the next person who has this problem.
I suggest that the problem may be from the primer, the stability of the primer was decreased due to damage that occurred for some reasons.
Colony PCR often gives false positives due to plasmid DNA still on the plate. I assume you re-streaked potential positives on a new plate and used those for colony PCR? If not, then go back and start by choosing other colonies.
You can't rush quality, good luck!
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