E-coli is eating up my plasmid! - (Sep/06/2006 )
please follow through the story if you have time. i have ligated and electroporated my plasmid into BL21 strain of E-coli (with which i had no problems before in preparing minipreps but with different plasmid), i got colonies check by PCR everything beautiful. I prepare miniprep, check OD DNA is there. i run the gel no band.. ..no matter how much i increase the volume no band. i run PCR on the minipreped plasmid the insert is there . so i think it must be BL21 and electroporate this DNA (minipreped from BL21) into JM109 strain. got a lot of colonies. PCR everything fine. do miniprep check OD everything fine. run the gel no band... . so i decide to do maxiprep on that. check OD it is aroung 2ug/ul.....run the gel no band!!! but when i run around 5ul of that i get a faint smear. so I guess my DNA is degraded. but my insert is there . please can you try to clarify this to me cause frankly i dont know what to do anymore.
Not an expert, but maybe some kind of nuclease contamination? Are you eluting your DNA in tris, water or TE? (maybe you should try TE as nucleases need ions that are chelated by EDTA).
Yes, I would also suggest you to use new TE for your DNA
just to b sure, check if the spectrophotometer (for OD) is working.
Use fresh buffers for the DNA preps.
just in case.. it even sounds funny. did you put ethidium bromide to the gel and/or turned on the UV light to visualize it?
Hahaha... sorry this isn't a funny matter...
So here is my try at solving the dissapearing DNA.
First, we must ascertain that there is DNA in your sample.
See if you can get a reading with the spectrophotometre with a sample that is known to contain DNA of known quantity. Do the readings telly?
Then get an OD reading of your sample. Is the DNA in there?
If there DNA is in the sample, then look at your equipment. Is your equipment the cause of the mysterious dissapearance?
Are your gel, gel buffer, gel box and loading buffer clean? How about using freshly made agarose and loading buffer and maybe wash out the gel box.
If the equipment is good, run a gel with your sample and a sample known to contain DNA. Also run an (5X and 10x) overloaded lane containing your sample.
If the lane is still blank, more so if the overloaded lanes show a smear, then unfortunately all your DNA has degraded.
Then you have to look at your preparation solutions and Ecoli. Somewhere DNAse has entered the system. Best to junk all your preparation solution at this point and start anew, also make sure nobody had a minor accident that they didn't tell you about and slipped in some E.coli which makes high levels of nucleases.
Best of luck
thanx a lot everyone! i had used controls with all the experiments, controls bands are always there but my sample has a smear. that is why i was sure that it is degraded. all i can think of is the BL21. since i have electroporated my original ligation mix into BL21, minipreped and electroporated this plasmid into JM109, i suspect i electroporated degraded plasmid by BL21 into new E_coli ...however if this is true i can't understand how the plasmid (if it is already degraded) got into E-coli. i always thought plasmids have to be circular to get into bacteria. and this supposedly degraded plasmid not only got into E-coli and gave a lot of colonies, but also these colonies ALL had my insert . but trying miniprep i never get DNA.
to tell you the truth the vector which is pHis from Santa Cruz have been tried with some students here and their ligations never worked. however they never had problems with getting enough DNA, they simply couldnt get the insert. So im thinking could my insert make this plasmid super unstable, so that it gets into E-coli but degrades during miniprep?!?! would like to hear your comments on that. please dont get bored with this it can get really interesting
You probably should make new loading dye, as well. The sucrose versions can grow things, which is very bad. We use ficoll based ones, which seem pretty immune to growth.
but i thought you did get a product of the right size on doing a pcr with the miniprep dna...so how can it be degraded...
I would use the term 'resistent.'. The ficoll version can also grow stuff, given time. I keep all the lab's loading buffer frozen when not in use.
EDIT: Just a thought, if the insert was causing instability, the typical thing would be to put it into either a BAC, a low copy or single copy plasmid. Another thing done, would be to grow the cells at 30 Celsius and process a large volume (in my lab typically from 500ml to 6L ) of this low density culture, to get the DNA
This might help... if the insert is the cause of your wows.