I already obtained two plasmid that harboring gene synthesis A or B. First is pUC57 harboring gene A (size of insert 1.5 kb). Second is pUC57 harboring gene B (size of insert 1.6 kb). Then, I transform these plasmid to E. coli DH5Alpha (Rubidiub chloride-Chemical competent cell). After get the transformants, I pick single colony of each plasmid, culture in LB+Amp 3 ml, Amp 100 ug/ml, 16 h and extract it by alkaline lysis method by using culture 1.5 ml or 3 ml. Finally, after ethanol precipitation step, I put 30 ul of TE buffer, and Run 2 ul on 0.8% agarose gel.
For pUC57-gene A is get a lot amount of plasmid. But for pUC57-gene B, I get very small amount of plasmid. I very confuse because I transform many time and pick many transformants to extract, but my result still the same. I doubt because difference only insert but the vector backbone still the same.
Could any one suggest me, how to disslove this problem?
Have you checked the growth of your E. coli transformed with vector B in comparison to A? Could it be that gene product B is toxic to your cells and therefore hindering growth or leading to lower copy number of the plasmid?
I guess the gene should not be expressed normally under your culturing conditions, but I think I have heard lacZ promotor is kind of leaky and might become a lot more leaky in stationary phase, maybe due to limiting nutrients (explained for example in this paper: http://onlinelibrary.wiley.com/doi/10.1002/bmb.25/pdf ). If that is the case, you might be able to see the effect if you compare the number of colonies formed from those stationary-phase-cultures.
Maybe going for lower culturing time (not going to stationary phase) might help, even if you are unable to see differences in growth in log-phase?
I don't know specifically about your vector backbone but, technically, if you incubate at toom temperature your columns 15-20 mins after washing with wash buffer to evaporate all of the ethanol (even after 2nd centrifugation), that'll increase the yield dramatically.
Are you 100% sure that your pUC57-gene B is what it's supposed to be? Are you getting enough DNA out to sequence it to make sure that it's the correct construct?
Dear Trivedi and Muhling,
Thank you for your suggestion.
I think may be sure because I order gene A and gene B synthesis from a company and a company already clone into pUC57 vector, and sequencing in the position of gene A and B.
And I get enough DNA for sequencing. I may be send it to sequence.
Have you checked the growth of your E. coli transformed with vector B in comparison to A? Could it be that gene product B is toxic to your cells and therefore hindering growth or leading to lower copy number of the plasmid?
I guess the gene should not be expressed normally under your culturing conditions, but I think I have heard lacZ promotor is kind of leaky and might become a lot more leaky in stationary phase, maybe due to limiting nutrients (explained for example in this paper: http://onlinelibrary.wiley.com/doi/10.1002/bmb.25/pdf ). If that is the case, you might be able to see the effect if you compare the number of colonies formed from those stationary-phase-cultures.
Maybe going for lower culturing time (not going to stationary phase) might help, even if you are unable to see differences in growth in log-phase?
Dear Winz,
Actually, gene A and B are a capsid protein of virus, but from difference species.
The capsid protein of this virus in nature cannot endanger anyone. So, I think that gene A and B also cannot endanger to E. coli.
And I observed that E. coli, harboring Gene A or B, can give a same amount of cell mass, but after plasmid extraction, the concentration of DNA is very difference.
Thank you for your suggestion.
hi
you can improve the yield of plasmid by incresing the culture volume to 5-10 ml. You should also increase the lysis reagents for complete lysis of biomass. Kindly include phenol chloroform extraction step to remove protein which can be detrimental for your future cloning work.
Dear Kumar, Khan, and Sandez,
Thank you for you suggestion.
I use Birnboim method for extraction a plasmid.
In my experience using Birnboim, i always use a 20 mL culture for plasmid extraction, 1-3 ml is too less material and you could be loosing it in some step. 20 ml culture extraction often yields 4000 or more ng/ul. I recomend checking your protocol and making sure you're following it correctly and not missing or doin some step wrong (losing the pellet or some precipitation).
I usually run colony PCR first to check correct transformation and then culture that colony for plasmid extraction. Check if your antibiotic is still working, if you are getting a lot of colonies they could be non transformed E. coli.
After getting the culture try to double pellet the culture before extracting the DNA using miniprep kit and dissolved normally in 50 ul at the end and in this case normally will have 150-300 ul of the DNA (because normally when using miniprep manually you will have more than 1000 ul of DNA) but because of purity we must use the kit to avoid having problems regarding the sequences late on
1. Are you sure the colony that you took is bacteria ? Some contaminating microorganisms produce colonies with a similar morphology and DNA extractions produce results similar tthat you have.
2. When you make extraction of DNA from a lot of material get low yields and dirty samples. Check if the amount of lysis buffer was added is in proportion to the biomass that you are lysed .Many bacteria does not mean much DNA actually means more problems. Everything should be in proportion .
Make a scanning spectrophotometer 200nm to 300nm . That will give you an idea about the type of contaminant being carried in the sample and tell you in the extraction step you were wrong .
If you are unsure of how to interpret the graph send me a picture and I'll help you solve it.
Dear Sandez, Al Shihi, Alonso, and Zaldastanishvili,
Thank you for your suggestion.
Sorry for reply late.
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Dear Alonso,
I'm quite sure that this bacteria is E. coli DH5alpha. Becasue I use the same stock of E.coli for transformation this plasmid and the other plasmid, but the problem still only happen with this plasmid. Althrough, I transformed the plasmid that have a problem many time, but every time and every transfomants still have low amount of plasmid DNA.
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