If you are willing to go "old fashioned" in non-kit labor intensive ways, then please consider phenol chloroform ether extractions. There are caveats including fresh equilibrated phenol, using a fume hood if possible, safe waste disposal, etc

If you are willing to go "old fashioned" in non-kit labor intensive ways, then please consider phenol chloroform ether extractions. There are caveats including fresh equilibrated phenol, using a fume hood if possible, safe waste disposal, etc

Dear Angelbert

• for extraction from samples with 'unusual' impurities like humic acid I concur with David and would elect to extract by phenol chloroform rather than newer silica based kit extraction kits
• Regarding protein contamination once again this is dealt by using proteinase K digestion as part of the traditional (manual) phenol chloroform extraction of genomic DNA:
• http://openwetware.org/wiki/DNA_extraction_from_tissue
• To remove contaminants from a pre existing semi purified sample I would simply subject to the same procedure again

Use phenol chloroform method used by Joseph Sambrook....For full details view the link below

http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4455.long 

For molecular biology applications, after step of DNA extraction, you can use QIAprep Spin Miniprep, for example.

Dear Angelbert

In view of the discussion thread and David's prior comments let me elaborate/ qualify a few things:

• For DNA Extraction use (as David says) fresh Tris buffered phenol: Tris buffered phenol is slightly alkaline and designed to extract genomic RNA. Do not confuse with Acidic phenol which selective enriches for and purified total RNA
• As David mentions fresh phenol is essential: Old phenol - that is phenol kept in the fridge for more than 3 months or especially just kept at room temp in the fume hood - will start to oxidise and turn brown: Many phenol preparations are yellow due to the presence of hydro quinones which act as 'sacrificial lambs' and preferentially oxidise in order to spare phenol: When these molecules have been consumes by oxidation phenol itself becomes the subject of oxidation and is converted to quinones. A sign of this happening is that the phenol begins to darken and turn brown. If your phenol begins to darken THROW AWAY (via a safe waste stream): Quinones aggressively degrade DNA and RNA !!!
• The best way to handle phenol is to freeze and the day before extraction thaw. At the end of the day prior to extraction shake vigorously to equilibrate: Equilibration ensures the phenol is the correct pH ensuring during extraction that DNA is partitioned to the upper phase and RNA is selectively sequestered to the interphase. It also ensures that the phenol is slightly water saturated: Unsaturated phenol is aggressively hygroscopic and sucks water from the upper aqueous phase during extraction which can compromise extraction
• When you digest your initial tissue sample in proteinase K plus SDS (In TNE buffer) 37C overnight with agitation will lead to better protein removal than 37C for 1 hour; Especially if you are having issues with protein contamination pre disposing to high 260/280 ratios > 2.0
• Better still  digest @ 55C overnight: 55C is normally advocated for short aggressive digestions (15 minutes to 1 hour) but prolonged incubation at this temp does not harm and in my experience might actually help for protein rich samples
• The following day add 1 x volume of your equilibrated phenol to your sample, VORTEX for 1 minute and spin at 13 K (if using 2ml screw cap tubes) or 5K in a sorval (for larger i.e. > 2ml extractions) for 15-20 minutes
• AGGRESSIVE VORTEXING AND PROLONGED SPINS lead to efficient removal of protein and discrete separation of the upper aqueous phase containing genomic DNA and salt from interphase RNA and coalesced lipids and some protein; to the lower organic phase containing primarily denatured protein. This helps with effective and 'clean decanting of the upper phase containing gDNA and primarily salt
• Remove upper aqueous phase FROM THE TOP and do not go all the way down to the interphase as this will result in interphase contamination (RNA, Lipid and protein & in your case perhaps humic acid)
• Now add this upper phase to an equal volume of phenol and chloroform and repeat the above: Ordinarily for small and/or clean sample sizes this intermediate phenol chloroform extraction might not be required but for samples with manifest protein contamination, i.e. 260/280 > 2.0 include
• Finally, perform a chloroform extraction (to remove residual organic material and also any remaining lipids, muco polysaccharides etc.) and precipitate by adding an equal volume of isopropanol and spinning as above: Perform this spin @ ROOM temp as refrigerated spins with isopropanol lead to selective precipitation and contamination of final gDNA with salt
• Remember after initial precipitation to perform a 70% wash and do this by adding 1-2mls of ROOM TEMP 70% ethanol and if pellet is visible vortexing: This facilitates desalting
• Finally spin again for 1 minute to secure pellet to the bottom of the tube; Remove liquid phase with a pipette; Spin AGAIN for a further minute to bring down residual ethanol and remove these last few drops using a P10 tip AGAINST the pellet
• This provisionally dries the pellet and allows full 'touch' drying prior to resuspension  simply leaving at the bench for ~ 15min
• DO NOT dry in a fume hood as laminar flow can detach the pellet and DO NOT vacuum dry as this desiccates the pellet and renders solubilising difficult 

I will finish by saying that for large sample volumes or complex materials consisting of large amounts of protein or lipid or humic acid (for example) It is better to perform this  initial manual extraction and then if you like mix your sample with an equal volume of 70% ethanol and column purify. I would not recommend direct extraction of lysates derived from tissue or complex materials as these can saturate and/or clog the column pores and either lead to impure samples owing to contaminant bleed through (and hence skewed 260/280 and 260/230 ratios - DO NOT FORGET the latter as < 1.0 indicates phenol contamination and/or salts) or failure to elute any DNA owing to column saturation   

Thank you very much I appreciate all your ideas, these will help me a lot. I'll get you back soon if the traditional method works in my samples. 

Great follow up details by Laurence. My questions are: 1. what is the intended use of your pDNA, cloning, in vitro expression, in vivo expression? 2. How did you determine that there was humic acid contamination? Humic acid is a collection of organic acids composed of carboxylates and phenols and is usually derived from microorganism metabolism of dead plants in soil.

To Laurence Stuart Hall: Thank you very much sir for these very overwhelming information. I would analyze first these things and familiarize the concept.s It seems that it takes time and days before having a pure DNA. 

I have a follow up questions, this is for purification right? So I will be processing my DNA sample already, not doing another extraction procedure? Thanks a lot. 

To Sean Sullivan: The community gDNA I extracted from the soil will be sent and analyzed through Next Generation Sequencing. I want to know the bacterial community structure of my sample and see its diversity.

I just thought that the possible contaminations if u have high A260/280 are proteins or humic acids, but proteins probably. My sample is taken from soil. Thanks 

Dear Engelbert

• Your question is actually the conclusion to my last answer
• With your PRE EXISTING SAMPLES YOU CAN CLEAN UP BY COLUMNS OR PHENOL CHLOROFORM plus ethanol precipitation as described below
• so let me elaborate:
• The last answer was a detailed synopsis of how you extract impure samples from scratch
• specifically prom K + SDS IN TNE buffer @ 55C over night followed by Tris saturated phenol/ phenol chloroform & chloroform extraction; followed by isopropanol precipitation and 70 % room temp ethanol wash is the most effective way to process lipid/protein and other impurity replete tissue/biological samples especially in larger quantities
• In contrast standard columns have limited saturation capacity and ability to deal with high lipid/mucoploydacharide or organic matter 
• However following initial removal of these substances by manual extraction supplementary clean up to inprove 260/280 ratio from pre purified samples can be done with columns:  this you could take existing samples with 269/280 > 2.0; mix with an equal volume of 70% ethanol and column purify:
• Your first column buffer having spun on your sample is designed to denature protein which is absorbed into the column matrix and retained when you elute your DNA.  If you elect to clean up your existing samples by going down the column route add your second ethanol based wash buffer; wait 1 minute spin then repeat this wash before spinning your column dry and then eluting genomic DNA
• Moreover to improve elution pre heat your water or TE to 50C; add; wait 5 minutes then spin elute
• Alternatively you can clean up your current samples by a stream lined version of the above 

Values of >2.0 on nanodrop are usually indicative of RNA contamination. Try an RNAse treatment of the DNA.. Just add RNAse (I was using a 200 ug/mL stock) to a final concentration of 20-50 ug/mL, incubate at 37C for 1 hour and then do a sodium acetate and isopropanol precipitation. You will lose some DNA, but it will be far less contaminated with RNA. If you need further information or are confused of the steps, I can send you the protocol I use.