Plasmid problems - low yeild plasmid DNA (Oct/18/2007 )
Hi there,
This is my first time on the forum but I will try to explain this as best I can for you...
I have problem extracting two plasmids...one with my gene of interest (HCV core) and the other is similar but without. The HCV core containing plasmid has been transformed (not by me) into E. coli DH10B and the other plasmid DH5A. I am not 100% sure what copy number is but have been told it should be medium.
I have tried numerous mini-preps, the Qiagen midi-prep kit and alkaline lysis with phenol/chlorform purification and only ever get back small quantities of plasmid DNA or none at all!! Only on one occasion I managed to yield 1mg/ml from a 500ml broth using the phenol/chloform protocol the one plasmid wouldnt not transfect...possibly due to genomic contamination
The plasmid has previously been prepared and transfectable
I have tried using different broth types, different amp conc, and the cells appear to be growing up fine
I have assessed the protocols step by step by taking out alliquots of the sample at various stages and can't find where the plasmid has disappeared too
There probably isn't that much there too begin with but I can't seem to get much back at all!
I have finished lab work atm but am curious to know why this problem may occur? could the host strain be spitting out the plasmid? What other explanations are there for this?
This is my first time on the forum but I will try to explain this as best I can for you...
I have problem extracting two plasmids...one with my gene of interest (HCV core) and the other is similar but without. The HCV core containing plasmid has been transformed (not by me) into E. coli DH10B and the other plasmid DH5A. I am not 100% sure what copy number is but have been told it should be medium.
I have tried numerous mini-preps, the Qiagen midi-prep kit and alkaline lysis with phenol/chlorform purification and only ever get back small quantities of plasmid DNA or none at all!! Only on one occasion I managed to yield 1mg/ml from a 500ml broth using the phenol/chloform protocol the one plasmid wouldnt not transfect...possibly due to genomic contamination
The plasmid has previously been prepared and transfectable
I have tried using different broth types, different amp conc, and the cells appear to be growing up fine
I have assessed the protocols step by step by taking out alliquots of the sample at various stages and can't find where the plasmid has disappeared too
There probably isn't that much there too begin with but I can't seem to get much back at all!
I have finished lab work atm but am curious to know why this problem may occur? could the host strain be spitting out the plasmid? What other explanations are there for this?
How long did you incubate your culture? What is the antibiotic concentration that you have used? Is the bacteria strain you used suitable for your plasmids?
This is my first time on the forum but I will try to explain this as best I can for you...
I have problem extracting two plasmids...one with my gene of interest (HCV core) and the other is similar but without. The HCV core containing plasmid has been transformed (not by me) into E. coli DH10B and the other plasmid DH5A. I am not 100% sure what copy number is but have been told it should be medium.
I have tried numerous mini-preps, the Qiagen midi-prep kit and alkaline lysis with phenol/chlorform purification and only ever get back small quantities of plasmid DNA or none at all!! Only on one occasion I managed to yield 1mg/ml from a 500ml broth using the phenol/chloform protocol the one plasmid wouldnt not transfect...possibly due to genomic contamination
The plasmid has previously been prepared and transfectable
I have tried using different broth types, different amp conc, and the cells appear to be growing up fine
I have assessed the protocols step by step by taking out alliquots of the sample at various stages and can't find where the plasmid has disappeared too
There probably isn't that much there too begin with but I can't seem to get much back at all!
I have finished lab work atm but am curious to know why this problem may occur? could the host strain be spitting out the plasmid? What other explanations are there for this?
How long did you incubate your culture? What is the antibiotic concentration that you have used? Is the bacteria strain you used suitable for your plasmids?
I have started from a ~8hr starter culture, diluted that into an overnight culture (25ml or 500ml depending on the prep) and incubated o/n.
I have tried innoculating from LB/amp plates and straight from glycerol stocks. I used 100ug/ml and 50ug/ml concentrations of amp. Not sure if the bacteria strain is suitable but it has worked before...
what temperature are you growing your cells? Try a lower temperature somewhere around 28 to 30 Celsius.
I would also suggest using a better medium. SOC can be used. It is expensive, but it is better then LB and can give better yield for difficult plasmids that won't behave. Also use a bevelled flask if possible. More oxygen means less stressful cells. Alternatively use a culture flask with a volume 10 times large then the medium volume. 50ml culture grown in a 500ml flask.
And finally there are two other trick you can try. One is to add chlroamphenicol to your culture several hours before you want to process teh culture. The chlroamphenicol will stop protein synthesis but plasmid replication continues. As a result plasmid yeilds increase.
A second trick you can do (if the cells are kicking out the plasmid- aka large cell pellet but no plasmid)... is to stop the culture early. Do not grow your cells to saturation. The culture won't be as dense so you will need to grow more flask to compensate, but the cells will probably contain the plasmid. See if you can programme your incubator. My Lab's sanyo incubator can be programmed, to drop the incubation temperature to 4 Celsius after a set duration of time.