First reduce the sample to powder or to a paste.

Vortex intensively and predigest the paste for 15 to 30 minutes with proteinase K in ATL buffer.

Inactivate proteinase K at 94°C for 5 MIN.

Vortex intensively your sample again..

Freeze it for 30 minutes.

Thaw the sample, vortex it and introduce 400 micoliters of the vortexed homogenate into the Magna Pure automatic DNA extractor.

Introduce before extraction an internal control(virus or plasmid) in the sample and in a second tube with the same volume of PBS to assess and validate the yield of DNA extracion of this procedure.

One simple method is to try a serial dilution of your extracted DNA (in sterile dnase and rnase free) water to dilute the inhibitors. You really only need a small amount of template DNA for a PCR and you can always rePCR your first product (not ideal). We used a 1:4 final dilution to reduce the inhibitors enough to get our PCRs to work (1 template:4 water). Try this, if it doesn't work let me know.

to reduce the PCR inhibitors in your extracted DNA

firstly you must use " inhibitory removal buffer" in ROCHE kits for DNA extraction and purification and then centrifugation.

second, during performing the PCR you must use the Tap polymerase contain glycerol which help in reducing the inhibitors in DNA extracted from bone.

best wishes

I agree tissue of any kind is extremely diificult to extract for DNA extraction .

Performing further serial dilutions would help.You only need a small amount of DNA for PCR .If 1:4 dilution does not work try 1:8 .

I hope this helps

Hi Pablo

What is the mechanism behind adding protease and freezing that removes inhibitors from bone?

Hi Catherine,

We alreday routinely do a 1:10 and 1:100 dilution of the extract. This usually solves the problem for tissue but bone is still usually inhibitory at 1:100. As 16s PCR is 10-100 times less sensistive that species specific PCR and the amount of bacteria in the sample is unknown I feel more dilution might just dilute any bacterial DNA into undetectability.

Hi Hosney

At the moment using a different Taq would require that we re-validate the whole assay which isn't a possibility at the moment. I'll look up the buffer you mentioned.

Thanks Valarie. See comments about dilution above.

Why you don't try to clean up your samples with magnetic beats ( prior your pcr). If you need I could provide the protocol

Thanks Marios. The MagNaPure Compact is a magnetic bead protocol already.

Hi Hannah,

I use the One STep PCR Inhibitor Removal Kit from ZYMO RESEARCH (www.zymoresearch.com) for my clinical bone-samples. The columns remove pcr inhibitors like melanin, polyphenoles, humic/fulvic acids, tannins etc, are easily to use and lowcost. Extract your DNA as usual and pipette the eluted DNA on the zymo column, centrifuge and use the supernatant for your pcr. For me it works very well with many kinds of problematic tissues/samples.

Good luck

Thanks Helana. If that kit works it it would be the kind of quick and easy process we could do as routine diagnostic. I have ordered a sample kit to play with!

Hi Hannah, you can try with a 2nd purification with PVP-TE (Lawson et al 1997). good luck.

I do agree with 2nd purification with PVP-TE

You may try a pre-treatment before following to DNA extraction by submitting your samples already grinded in 0,5 M EDTA, pH 8,5 solution for 48h. After that time remove the solution containing the EDTA. EDTA will demineralize or will capture bi-valent ions such as Ca++ and MgCl++ in excess that inhibit the PCR.

To be honest I don't like the look of the PVP-TE procedure because it involves preciptation and a lot of hands on time. Fine for research but I'm designing things for a busy diagnostic lab where things need to be easy, fast and perferably automatable with ready made reagents. Kits work out cheaper than paying people to do fiddly procedures and are much less error prone which is vital when working with clinical samples.

I will however store the idea away for any pure research projects in future!

Thanks Laura. I can't add 48 hours to a diagnostic test! However, I'll look into perhaps another way of capturing excess ions.

24h may also work if possible!

Half a working day would be the longest possible addition to the protocol and only if it was mostly "hands off".

Hannah, it may be worth trying an engineered polymerase, such as: http://nar.oxfordjournals.org/content/early/2011/02/04/nar.gkq1360.short

Its not commercially available but expresses easily and you can probably get a sample for testing (disclaimer: I currently work for Phil Holliger but had nothing to do with this project, and in any case requests for polymerases would have to go via him).

We are using Qiagen DNA extraction kits for tissue and blood and it is works perfect. Price is different but quality much better.

You can find details here: http://www.qiagen.com/Products/Catalog/Sample-Technologies/DNA-Sample-Technologies/Genomic-DNA/Gentra-Puregene-Tissue-Kit#orderinginformation

http://www.qiagen.com/Products/Catalog/Automated-Solutions/Sample-Prep/EZ1-DNA-Tissue-Kit#orderinginformation

http://www.qiagen.com/Search.aspx?q=DNA%2520from%2520tissue#&&p=1

Hi Giorgi. I'm interested that you say Qiagen gives you better results. I did consider using the EZ1 as it offers an automated platform with single use reagent packs. Have you got any data on how it is better quality? What protocol card would you use on the EZ1 with the tissue kit? Do you use the DNA tissue kit for all sample types?

Hi Hannah,

I have had a same problems with bone marrow. We are working with QIAGEN since 1997 and it`s works good. I use different types of DNA kit. We use Tissue and blood for those samples only, stool DNA kit for stool materials for intestinal pathogens and the regular DNA mini or MIDI kit for all kind of bacterial samples from cultures. Again in case of bone marrow what I am doing is to leave the small amount (one or two drops) of bone marrow sample through the night in ATL provided with QIAGEN DNA tissue and blood kit. In ATL lysis is very good. Unfortunately I can not give advice regarding the EZ1 as far as we do not use automated platforms. However I know from my colleagues from United States CDC that EZ1 is very nice too. One more trick, try to use Protease K instead of Proteinase K.

Hi Hannah, I agree with you respecto to PVP-TE, if it´s for diagnostic is no useful. Perhaps you use Expand Long DNA polimerase for your PCRs, this polimerase woks very well with inhibotories. good luck.

Hi Hannah,

You may try 5' TINA modified primers (e.g. from Trilink: http://www.trilinkbiotech.com/products/oligo/synthesis/TINA.asp) - no need to re-design the primer sequences - just order with the TINA molecule as an add-on at the 5' position

This modification increases PCR efficiency in cases with difficult-to-test samples - e.g. soil, feces - and also bone, I would guess.