4 replies to this topic

[angel0071987]

HI
i was wondering if anyone could suggest ways to remove carbohydrate contamination from DNA preparations. I am using oragene kits to collect saliva and extract the DNA (from dogs). the extraction process is basically just ethanol precipitation of DNA. The problem im having is that the DNA has loads of carbohydrate contamination in. I think the high contamination levels are due to owners feeding the dogs just before collecting sample!!
I have tried etanol precipitation clean up (to no avail)
i am going to try chloroform cleanup - does anyone have any tried and tested methods for this?
does anyone know of any other methods i could use columns or the like?
The DNA yield itself is not huge, so aany techniques that minimise DNA loss would be great.

i have been told that the carbohydrates will not affect any downstream application i want to do GWAS and sequenom to name a few, but i would like to try and clean up the DNA.

THANKS IN ADVANCE

[HomeBrew]

Hi angel0071987 -- welcome to the BioForums!

Ethanol precipitation will (as you've found) also recover carbohydrates -- not just from food, but also from bacteria in the sample and from the somatic cells.  There are spin column kits that will recover just the DNA (from Qiagen perhaps -- I'm not familiar with recovering DNA from saliva; perhaps they have a kit specialized for saliva).

What DNA are you interested in -- thye dog's DNA or DNA from oral bacteria?

You could also clean up the carbohydrates with a phenol:chloroform extraction followed by ethanol precipitation.

[mdfenko]

norgen biotek has a saliva dna isolation kit.

(it says for as little as 100ml saliva but i think they meant to say 100ul, the specifications say that 0.5ml is the maximum amount of saliva for extraction)

Edited by mdfenko, 08 October 2010 - 07:10 AM.

[angel0071987]

the dogs DNA is the one im interested in. Hopefully the oragene kits im using stop bacterial growth as soon as the sponges are placed in the preservation solution in the tube. it says in  the documentation that less than 16% bacterial contamination is expected, so the majority of DNA is canine!
I will investigate the columns suggested. Thanks very much!

[chromatin]

Pass them through columns such as qiagen columns. it should work.
http://www.bioprotocols.info