I used PCR to add a few nucleotides to the 3' end of a gene on a pc3.1 plasmid and produced a linear vector with 18bp overlap at 5' and 3'. Then I used DpnI to cut off the template chain, transformed the linearized vector into BL21, plated it, picked a single clone and continued to culture it in Kan-resistant LB culture medium, and then handed it over to a sequencing company. The sequencing company said that my bacteria did not grow when further expanded and could not be sequenced. So I extracted the plasmid and sent it for sequencing again, and it still showed no signal. I simply asked the sequencing company to continue testing the Kan resistance gene, and it still showed no signal. If it is because the antibiotics are degraded, then when I did the transformation, the negative control that did not transform any genes did not have any colonies, which shows that the antibiotics worked. I am very confused. If the plasmid was not successfully transformed, why can it grow in the resistance culture dish?
BL21 is not a good strain to propagate plasmids (they are for protein expression); use DH5alpha, make minipreps and analyse the plasmids with restriction enzymes / gel electrophoresis before to send them to sequencing
Sounds like you'll need to start over from the PCR step.
One colony on a plate can be contamination with another species of bacteria or another strain of your bacteria (anyone else using kan resistance in your lab?). Also, mutations that allow antibiotic resistance happen at a low, but not 0 level.
Remember, your goal is to make the plasmid as part of a bigger project, not to chase down exactly what went wrong.
Good luck!
Sometimes it grows even when was not transformed, that happened quite often. Collect a part of your colonies with a toothpick, boil in 50 ul of MQ water for 5 minutes and use 0.5 to 4 ul of this boiled to amplify your gene of interest. Track the positive one back to your plate.
I completely agree with the previous responses. I've also encountered the issue of non-specific single colonies on transformed plates, which is why I never rely on just one colony for sequencing. I typically pick several colonies (at least 10-15, depending on the transformation efficiency) and perform a colony PCR before sending them off for sequencing.
Also as Didier already pointed out, BL21 cells are not the best for propagating so sometimes It might also not maintain the plasmid at high copy numbers in which case the sequencing depth can be increased. Overall there might be several other factors like plasmid instability, self ligation etc.
So for me at the end, starting over would sound more efficient than trying to troubleshoot exactly what was wrong, as Katie mentioned.
Hope this helps, and good luck!
When plasmid sequencing yields no signal, it's often due to a range of issues such as low-quality or insufficient DNA, primer problems, PCR optimization needs, sequencing reaction flaws, instrumentation or software glitches, complex plasmid structures, contamination, expired reagents, or simple human mistakes. To tackle this, researchers must refine their DNA preparation and PCR protocols, explore alternative primers or sequencing approaches, double-check instrument and software configurations, and conduct diagnostic tests with positive controls. By methodically evaluating and addressing these potential factors, scientists can surmount the obstacles of no signal in plasmid sequencing and achieve precise, trustworthy outcomes.
It could be a contaminating bacteria that integrates the plasmid into its chromosome, so you cannot obtain any plasmid. I would start over transforming DH5a.
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