We have a plasmid construct that either cannot be extracted by miniprep methods, or if extracted, only in tiny amounts. The backbone of this construct is PUC19. Any suggestions to overcome this extraction problem?
You need to check the strain that you are using. DH5a/JM109 should work fine if the plasmid has pUC19 backbone. Use the fresh culture for transformation and it should work fine.
Also try to change the solutions that you are using for the extraction. Prepare fresh solutions and extract the plasmid.
Dear Shivakumara
thanks for your answer
We use 3 different strain, fresh culture and 3 methods for plasmid extraction but we could not obtain any suitable thing
Mostafa, your plasmid might be toxic to the bacteria. If you look at the broth culture, do you see any anomalies such as low cell density even after extended culture times or long strings of cells? If you can, examine the bacteria under the microscope too.
You may have to resort to extra ampicillin or harvesting more cells to increase your yield.
You mean you have purified other plasmids in parallel and they work fine? And this is the only one where the standard preps fail?
What is within this construct? Does it have long repeats? Or does it contain the coding sequence for protein of some sort?
For unstable plasmids such as those containing repeats or high GC content, you can try growing them at 32C for longer periods of time. The alternative is to take existing plasmid stock and transform into bacteria strains which are specially made to grow unstable plasmids.
I presume you are streaking some sort of glycerol stock on a plate prior to starting the liquid culture?
Finally, what concentrations are you getting from your minipreps?
Dear Mark
Thanks a lot
It grows slower than untransformed DH5a bacteria
I use 10 ml bacteria culture, but it be exteracted ina very low amount in compared to another plasmids
Dear Alejandro
thanks a lot
yes. we exteracted another plasmid in parallel and it was very good in cocentration but this one cannot be exteracted by same method
Dear Brian
thanks a lot
we have a fused gene that is "plyGBS" and on the electrophoresis gel we see a thin band but by biophotometere we read between 0.5 t0 1 micro-gram per micro-liter.
Dear Mostafa,
I do not know if you are using any Kit for minipreps or sone other method.
I was also dealing with PUC19 during my PhD work and I was using "GeneJET Plasmid Miniprep Kit" from Thermo Scientific and it was working really great.
Besides this kit, I was using an other method which we called "Rapid Boiling method for Plasmid Isolation with large quantity but rather less quality. this method is given in the following link.
http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/Plasmid_Isolation_from_Bacteria.pdf
Moreover, You can try DH10B strain for transformation and Plasmid Isolation.
Best Wishes,
Hi Mostafa, a quick search reveals that plyGBS is a proteolytic enzyme which is lethal to bacteria, or at least harmful. That tells me that your low yield is most likely because of expression of this toxic enzyme.
puc19 is a very high copy number plasmid, so leaky expression of this toxic gene is quite probable. I can suggest 3 approaches:
1) use a tighter control bactera strain, such as lucigens high control BL21 or 10G cells, or any other cell type designed for toxic gene propagation (they generally use inducible systems such as arabinose for control)
2) use a lower copy number plasmid to lower the chances of leaky expression.
3) Cheapest but least likely method, is to simply grow at room temp and hope that expression is lessened.
on a side note, i assume your concentration you said is a typo? .5-1 ug/ul is a ridiculous amount of dna from a miniprep. .5-1 ng/ul on the other hand is most likely what you meant?
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