Plasmid DNA extraction problem - (Dec/23/2007 )
after electrophoresis,nothing could be found in agarose,
the bacteria could grow on the resistance plate, the transduction should be sucessful,
how to explain it?
What was the extraction procedure you followed? Are you sure you got that right?
quite a complicate question.
We need more information about the cell strain that you are using, the copy number of your plasmid, and the nature of insert that said plasmid is carrying. Also any information about the growing procedure and what you actually did during the plasmid extraction can help provide clues to what may have happened.
1- your antibiotic plates could be old, thus the anything grows even cells which do not have the desired plasmid. Alternative might the antibiotics in the culture medium that you use to grow the cells in be old?
2- Might your insert be unstable, and thus lost when cells are stressed. In which case growing your cells in a rich medium (eg SOC or Terrific Broth) at a lower temperature (28 Celsius to 30 Celsius) could help in a such a situation. If things are rather bad, grow your cells only to mid log phase and no futher.
3- You are using an expression strain to grow a plasmid which has a leaky promoter... resulting in low level expression of a toxic protein. Drop the temperature, and harvest cells early. If that doesn't work change to a tighter promoter
4- You are growing a very low copy plasmid. You thus need to grow a larger volume of cells to harverst said plasmid.
5- You plasmid DNA is being degraded, this can be due to using an expression or wildtype strain for growing your plasmid. Such strains tend to have functional endonucleases, which degrade plasmid DNA when the cells lyse. Change to cloning strains, they were made for cloning so use them. Alternatively your alkaline lysis solution have become severely contaminate. Change/make up new solution
6- Your DNA binding column, is binding all your DNA and won't let it go. Try heating your elution solution to 65 Celsius prior to elution
7-Your DNA binding column is not binding any DNA... is the column old? Have you checked the expiry date? Did you add isopropanol to the DNA solution prior to running said solution through the column?
8- Could this be a technical error? Is anybody else in the lab suffering similar problems. Has somebody in the lab switched solution without telling anyone. Was the phenol/chloroform solution made up right? (if you are using it)
9- You loading dye/buffer could have become contaminated. All your samples are thus degrading on the gel whist running.
etc etc... Just an example of what could have gone wrong. As you can see, many things can cause your DNA to dissapear, or not there to begin with. More infomation of what was actually done, every detail that you can remember.
We need more information about the cell strain that you are using, the copy number of your plasmid, and the nature of insert that said plasmid is carrying. Also any information about the growing procedure and what you actually did during the plasmid extraction can help provide clues to what may have happened.
1- your antibiotic plates could be old, thus the anything grows even cells which do not have the desired plasmid. Alternative might the antibiotics in the culture medium that you use to grow the cells in be old?
2- Might your insert be unstable, and thus lost when cells are stressed. In which case growing your cells in a rich medium (eg SOC or Terrific Broth) at a lower temperature (28 Celsius to 30 Celsius) could help in a such a situation. If things are rather bad, grow your cells only to mid log phase and no futher.
3- You are using an expression strain to grow a plasmid which has a leaky promoter... resulting in low level expression of a toxic protein. Drop the temperature, and harvest cells early. If that doesn't work change to a tighter promoter
4- You are growing a very low copy plasmid. You thus need to grow a larger volume of cells to harverst said plasmid.
5- You plasmid DNA is being degraded, this can be due to using an expression or wildtype strain for growing your plasmid. Such strains tend to have functional endonucleases, which degrade plasmid DNA when the cells lyse. Change to cloning strains, they were made for cloning so use them. Alternatively your alkaline lysis solution have become severely contaminate. Change/make up new solution
6- Your DNA binding column, is binding all your DNA and won't let it go. Try heating your elution solution to 65 Celsius prior to elution
7-Your DNA binding column is not binding any DNA... is the column old? Have you checked the expiry date? Did you add isopropanol to the DNA solution prior to running said solution through the column?
8- Could this be a technical error? Is anybody else in the lab suffering similar problems. Has somebody in the lab switched solution without telling anyone. Was the phenol/chloroform solution made up right? (if you are using it)
9- You loading dye/buffer could have become contaminated. All your samples are thus degrading on the gel whist running.
etc etc... Just an example of what could have gone wrong. As you can see, many things can cause your DNA to dissapear, or not there to begin with. More infomation of what was actually done, every detail that you can remember.
thank you for your so detailed ananlysis on the problem.