OD ratio of 1.9 to 2.2 sounds more like RNA or dna with some chemical contamination aas dna should be a maximum of 1.8 so it may be that your sample is rna,bacterial dna and a small amount of plasmid dna. Genomic dna will be a smear so restriction cutting will just produce a smaller smear and once dna is smeared over the length of the gel it is much fainter and hard to see. Are you seeing your size standard (ladder) clearly and can you attach a picture of the gel?.Are you looking for natural plasmids or are these plasmids that you have put in the bacteria?

OD ratio of 1.9 to 2.2 sounds more like RNA or dna with some chemical contamination aas dna should be a maximum of 1.8 so it may be that your sample is rna,bacterial dna and a small amount of plasmid dna. Genomic dna will be a smear so restriction cutting will just produce a smaller smear and once dna is smeared over the length of the gel it is much fainter and hard to see. Are you seeing your size standard (ladder) clearly and can you attach a picture of the gel?.Are you looking for natural plasmids or are these plasmids that you have put in the bacteria?

Also are these bacteria you are able to grow in culture? If it's an issue with not enough DNA then growing them for longer would help. But as Paul said a picture would also help. Can you see anything at all? Depending on what your gel looks like it might help to work out if it's a sample issue or an issue with your gel/running set up.

I agree with the previous commentors a picture would be helpful. Also if you concentration truely is 34.2ug/uL and not 34.2ng/uL then 1uL will more than show up on a gel. Even with a very broad smear usually 1ug is enough to show up. With RNA as well though usually 1ug is enough to see the rRNA. So I would consider if your concentration truely is 34.2ug/uL.

I agree with the previous commentors. It would be helpful if you can share the gel image with us. Can I also ask what you need to use DNA for? If you can culture the bacteria then that would be a good option to increase your DNA concentration.

Doing PCR might help to you to know if the DNA is actually there. Paul Rutland mentioned that there might be RNA and some contamination. PCR will help you say that DNA is present. Just make sure you dilute you DNA 1:10 and maybe 1:100 before doing PCR to reduce/eliminate the effects of contamination on PCR.

Let us know how this goes.

The original post specifically mentions plasmid DNA. Are you trying to get plasmid DNA or genomic DNA? I would note that if you are using a plasmid extraction kit then you are probably discarding most of the genomic DNA but natural isolates may or may not have plasmids and in most cases the plasmid are quite low copy number, often quite large, and may prove difficult to see for those reasons. Since they are large, they shear a lot and with low copy number you won't recover very much.

Hi Kitt,

I would suggest the PCR product as a DNA template in order to get a high concentrated PCR product. I think after this step you can get your target.

Paul Rutland The ladder appeared fine, but the other lanes were completely blank.

I'm not necessarily looking for anything in particular; I took some swabs from my pet chinchilla and wanted to find out what kind of bacteria is growing on him. I don't have a ton of experience with this kind of microbiology work, so I figured in my free time I'd use some of the leftover/unused supplies we have at work to experiment and become more comfortable/understanding of these processes. I'm kind of winging it right now.

Hi Kitt Kroeger , what Michael J. Benedik suggested seems to be true in your case. As you mentioned now, you are looking for what kind of bacteria is growing on a chinchilla, I would suspect those bacteria won't have or very less plasmid. In a very general sense, the plasmid is not required by bacteria for its survival and so it is also called extrachromosomal DNA. In our lab work, the bacteria contain antibiotic resistance genes in their plasmid but, if you culture the same bacteria in a normal plate without antibiotics, the bacteria will soon lose the plasmid as it doesn't need for its survival.

The above explanation I gave because in your study there is no antibiotic selection going on and thus, we would expect negligible plasmids. If you are interested to get both bacterial chromosomal DNA and plasmid then it is better to do a manual DNA extraction protocol without any kit that will give a good amount of DNA.

On a related note, as your study is to look for what are the different bacteria then it is better to collect the 16s ribosomal RNA.

Hi Kitt Kroeger ,

Sounds good but we have a problem with amounts here. Swabs will not produce visible amounts of dna. There are just not enough cells so I think that either the nanodrop was wrongly zeroed/blanked...it must be done with exactly the same solvent as the dna is dissolved in. Restriction digest may have worked but mouth dna is often randomly cut and enzyme degraded so digesting will simply give a slightly smaller smear of dna and the smaller dna will be spread over the length of the gel and at any one position will be too faint to see. Pcr product is visible because it is all the same size and so is very bright as there are millions of copies of the product. Each 10 pcr cycles is 1000x more dna of that size. One cell contains about 6picogrammes of dna so it will take many millions of cells to get a decent yield of visible dna but you will have plenty to run a few pcrs which, if they work, proves the existence of the dna that you have made. Onions are a good source of quite protein free dna if you want to make visible amounts just to see what it looks like