I have been regularly amplifying bacteria with my plasmid of interest and performing midipreps yielding 400-600 ng/uL. As of late, my yields have been very low tanking to almost 80 ng/uL. I initially thought this may be due to antibiotic degradation so both fresh LB and ampicillin were made (working concentration of 50ug/uL). This actually decreased the yields.
I have also noticed a decrease in my transfection efficiency. May this be due to the diluted plasmid yields increasing the volume added to the wells? Any tips on how to resolve this?
Best
Incubate the bacteria for more time and provide proper aeration , also the decrease in transfection efficiency could be due to plasmid concerntration being less or the if the cells are not competant enough , try to increase the plasmid concerntration and elute the plasmid in less elution buffer so it doesnot become diluted , also heat the elution buffer to 37 or room temperature, it gives a good yeild.
Hi Douglas Se
I see your plasmid yield has dropped, but has your cell mass dropped? If the weight of cell pellet dropped, it could be because of bacteriophage contamination, in which case you need to autoclave pipettes in addition to other precautions.
Now, if your pellet mass is roughly same, you can incubate bacteria for more time with another dose of antibiotic after 16hrs incubation. This will keep the bacteria under pressure for plasmid maintenance.
Hope it helps,
Subham.
Most likely the problem falls into one of these categories
1) not achieving high cell density (problem with medium or antibiotics or phage contamination)
2) Cells grow but have lost plasmid (can easily test this)
3) problem with the midipreps not working as well as before (bad kit, problem with reagents, etc).
I would carefully check each of these and then you can figure out what is the source of the problem and an appropriate solution.
- Badges
- Science topic