Low concentration DNA, quantity needed and any ideas?!

[firefly2280]

Hi all

I was wondering if anyone had any experience of low concentration DNA and if there was a method to improve extraction?

I am currently taking environmental swabs of quite a small area (which can not be made larger) and doing a bead-beat and Qiagen column extraction.

I am getting around 6ng/ul showing on a Nanodrop and nothing coming up on an endpoint 16S PCR

I was wondering how much starting material you might need to be able to feed into the MiSeq?

Thanks for your help!

nikki

[yaximik]

6 ng/ul is a pretty good amount, but Nanodrop is not the best method as aptical density can not discriminate between DNA and nucleotides, for example. I would use Nanodrop 3300, people use Qubit, or anything that is suitable for fluorescence. Once libraries are made, these can be carefully reamplified to load MiSeq properly.

[firefly2280]

Thanks, yes I know nanodrop isnt the best, it was just to get a rough idea.

But you think this should be enough despite not seeing a band on a gel after an endpoint PCR?

[pbluescript]

I don't trust anything lower than about 10 ng/ul from a Nanodrop. You can put water on there and get a reading of 6ng/ul. You should use a fluorescent dye to quantify instead.

[protist]

Completely agree with pbluescript and in fact I personally would change that limit to 15-20 ng/uL. Nanodrop is good for 260/280 and 260/230 ratios but for accurate quantification at the lower 10 ng range you really should use Qubit or some other fluorescent based methodology.

[firefly2280]

thanks for all of your replies

My main question really is what can I do to increase my yield of DNA as I clearly dont have enough?

thanks

[HESmith]

Forget quantification. Given that PCR can detect even a single copy of template, my concern would be that you have virtually no DNA in your sample. I would recommend reviewing the literature for techniques appropriate for DNA isolation from small amounts of material. I'm fairly certain that beadbeater and Qiagen columns will not be at the top of the list...

[protist]

thanks for all of your replies

My main question really is what can I do to increase my yield of DNA as I clearly dont have enough?

thanks

You don't say how much DNA you actually have or the total volume only the ng/uL There a several kits out there for low starting amounts eg Nextera which is recommended for MiSeq..

Nextera XT kits from Illumina - libraries can be constructed from as little as 1 ng of starting material

Nextera Kit Illumina/EpicentreBio - libraries from a 50 ng and up..

[protist]

Might be of help..

http://www.biotechniques.com/news/DNA-Extraction-Overcoming-Obstacles-in-Microbial-Studies/biotechniques-335698.html?utm_source=BioTechniques+Newsletters+%26+e-Alerts&utm_campaign=6b08ec969c-sequencing-newsletter&utm_medium=email#.UIgm3kTgK2w

[pbluescript]

thanks for all of your replies

My main question really is what can I do to increase my yield of DNA as I clearly dont have enough?

thanks

I routinely work with small amounts of RNA and I had to abandon the use of columns for purification due to sample loss. Mostly, I use organic methods, precipitation, and Ampure beads for isolation and purification.

[rnaeye]

Environmental samples may contain PCR inhibitors. If your A260 reading is coming from nucleic acids, it's good amount of DNA to do PCR. You can run ~10-20 ng DNA on a garose gel and confirm that you have DNA. To me your problem might be PCR inhibition.5-10 ng would be enough to make library.

[pmiguel]

I am with Harold here. Nanodrop is not only useless at this concentration, it is likely to give you and incorrect result even at 10x this concentration. This is because genomic DNA preps generally will contain large amounts of RNA (more than the DNA in the prep.) So what is the point of checking absorbance at 260 nm as it does not distinguish between RNA and DNA?

This does not even get into other contaminants that frequently adulterate DNA preps. Run a gel or use a fluorimeter.

--
Phillip

[firefly2280]

Thanks for your help all.

Nothing shows up on a gel, im quite sure there are inhibitors, which is why i wanted to use a column based extraction method. I might switch back to phenol chloroform if thats better, although when I compared it with a couple of kits and did a qPCR, my curves and replicates were much nicer with the qiagen.

I will check out the Nextera kits too and I have some picogreen to check my concentration.