I have a bacteria and upon SI nuclease PFGE I came to know that it contains 2 plasmids (140 k and 350 kb respectively). I am interested to isolate one of the plasmids. Once I tried to isolate the plasmids and upon normal gel electrophoresis I found the plasmid are not moving to their expected size, and are gathering at the same level. I am thinking the plasmids are circular/supercoiled!!
May I request you to suggest me a comprehensive protocol to differentiate the plasmids in Gel or any other way and have a good quality plasmid!!
Thanking you,
Jayedul Hassan
You are right, circular plasmids tend to assume higher order structures and do not always line up to the expected molecular size.
Do you know what plasmids they are? If you know it, linearise the plasmid using any suitable restriction enzyme and then it should line up exactly at the molecular size of the plasmid. Then you can perform a gel extraction.
Dear Girish,
Make plasmid separation in low % of agarose (app. 0,5%) in two parallel wells. Cut the gel after migration into 2 pieces. Use one for staining and estimation the migration distance with UV. Mark the position of the bands in the second line. Just below of each band (probably you will have 4 bands, respectively for CCC and OC forms of each megaplasmid) cut the gel and put a small dialysis bag containing the buffer with EDTA and opened towards plasmid bands migration. The same make with stained portion (reference). Switch on electrophoresis and run gels in parallel for a few minutes. You can control the migration by checking the reference. As far as your reference DNA (first one of 150 kb) enters the bag, stop electrophoresis and collect the non stained DNA from the bag. Continue electrophoresis in the same way to get 350 kb plasmid. Manipulations must be done very carefully, in the absence of nuclease contamination. Migration should be done at low voltage to diminish DNA nicking.
Best wishes
Actually, I don't have enough idea about the plasmid and its restriction sites. This is a very new report. I found that my gene of interest is located in this plasmid. I have the gene sequence but there is no restriction endonuclease site in this gene. What would e the best restriction endonuclease in this aspect? @Girish Kumar Kaitholil
I don't have enough knowledge on electrophoresis using dialysis membrane as you mentioned. May I have more detailed information regarding this? @ Vehary Sakayan
Thank you very much for your responses.
Dear Jayedul,
You mean that there is no suitable site? If you know the genus of your bacterium, look the NCBI site for the sequenced genome close to your strain. Probably, similar plasmid has been sequenced and the restriction map is available. Otherwise, check the GC composition, it might help you in choosing the most appropriate enzyme.
Search for "preparation of dialysis membrane for DNA purification" in internet. With your experience in PFGE you will be able to make the plasmid purification.
Finally, if you know your gene's sequence, why not using PCR for amplification? More simple way for cloning and further analysis of the plasmid of your interest.
Best wishes
Dear Vehary Sakanyan,
Thank you very much for your nice suggestions.
I have the sequence of my gene of interest. I did already reveal the important features of this gene. While I was trying to find out the location of the gene in the genome, I found that it is located in a large plasmid (As I mentioned earlier, my isolates have 2 plasmids).
My isolate belongs to Providencia spp. As far I know, there is only one whole genome sequence available in NCBI site and no information about such plasmids and gene!!
Now, I would like to do some research about its mechanism of transfer (horizontally transmissible or not) and some other features. For this purpose, it would be nice if I could isolate the plasmid in pure. Thus, I am trying to isolate the plasmid in pure. But, I can not overcome the contamination from the genomic DNA and the other plasmid.
What is your suggestion in this aspect?
Thank you very much for your suggestions again.
Sincerely,
Jayedul Hassan
If I understood the question, you have already separated both plasmids by PFGE and gel-purified them, and are asking for advice in how to tell if they are only different topoforms of the same plasmid or different plasmids altogether, right? In that case, the simplest solution would be to pick several different restriction endonucleases (6-bp site cutters preferably) and compare the restriction patterns of both isolates. Or, you could treat them with topoisomerase I (I think NEB sells it) to see if they are supercoiled and nicked versions of the same plasmid. Or, if the sample is too small, you can instead dot-blot both samples and probe them with a labelled fragment of your gene to see which one is hosting it, and then work from there.
Hope it helps!
- Badges
- Science topic