Hello Cintia,

I have only ever used commercial kits to extract DNA from agarose gels (such as those made by Macherey Nagel and QIAgen) and I have never heard of the method you are describing.

It seems to me, that the risk of contamination would be great and I would expect the extraction efficiency to be low and the product impure. Without any reagents or charged column to manipulate the gel matrix, I cannot see how simple centrifugation would efficiently extract the DNA. What is the purity of your obtained product? (if any)

Where does the strong positive band of your negative control occur - at the predicted size of your product? If not, my guess is that it might be a result of primer dimerization or an amplified non-specific product from your first round of PCR.

Hello Jeppe,

thank you for your answer! I get the band in my negative at the expected size (around 500bp), but still weird that it was always negative in the first steps with the same polymerase after PCR. This is why I think it is because of this gel extraction method. I don´t even know how I could cite this without literature. Purity is quite bad of course. The basic idea here was that the filter tip only allows molecules under a certain size to go through.

Hello again,

The only thing I could find about the method seems to be this news article from 2016:

https://www.genengnews.com/gen-articles/heres-an-inside-tip-for-dna-extraction/5834

Apparently a firm has developed special pipette tips with smart surfaces, that are able to bind DNA. However, I would doubt the results without the special pipette tips.

Of course there will be DNA in your eluded product, but my guess is, that if you liquified your excized gel band, you would achieve about the same purity. I suspect you need very special tips for this method to work based only on size exclusion. If you find a way to make it work, and there is no scientific litterature about the method, I do not think you need to cite it at all; it should be a novel method developed by you, perhaps inspired by some source.

Is there any way, that you might have contaminated the negative control? Gel loading difficulties, forgetting to change tips, etc.? This might explain a veeeery low amount of product in the first round, but a significant amount of product in your second round.

Hi,

I just got this from a colleague: https://openwetware.org/wiki/User:Andrew_Perry/Protocols/DNA_gel_extraction

it seems that some people are actually using this.

Hello,

Alright, nice, exciting stuff - you even got a cited source as well. Best of luck with it!

Dear Traci L Testerman ,

thank you for your answer and tips! I was familiar with the buffer problem so used TAE and for gel not ethidium bromide but Midori Green.

Hello Cintia,

interesting protocol, never heard of it.

you should conduct a negative control consisting of a flow through of a pipette tip with solubilisation medium only. If your band appears again, your tips are (most likely) contaminated with DNA from whatever source. You could also try the same control with a freshly unwrapped box of filter tips. And of course it could be possible that any other component (vials, buffers) are contaminated. Did you sequence the "non-specific" band? If you use primers designed to amplificate 16S of a multitude of species , it is highly likely that any bacterial contamination will be amplified, too. However, I would recommend a commercial column based purification kit, as Jeppe Hedegaard Kallesøe mentioned above. It will save time and should give good results.