Dear all, I am following a gDNA rapid salt-extraction method for PCR-based techniques reported in Nucleic Acid Research Journal. Here is the protocol:
100mg minced meat homogenized in 440 µL TNES buffer ( 0.4M NaCl, 10mM TrisCl, 2 mM EDTA, 2% SDS). Add in 400 µg proteinase K and mixed well. Incubate the mixture at 55°C overnight. Add 300 µL 6M NaCl, vortex for 30s and centrifuge at 10,000 xg 30 min. Supernatant transfer to new tube, add equal volume of isopropanol and incubate -20°C for 1h. Spin at 10,000 xg 20 min at 4°C. Wash with 70% EtOH, resuspend the pellet in 300 µL ddH2O.
Nanodrop showed me DNA with concentration 138ng/µL. A260/280= 1.85 A260/230=1.2 ( A230=0.055, A260 =0.065, A280=0.035). About 690ng is screened on 2% agarose (attached- first lane). Then I make a 10-fold serial dilutions start from 100ng but sadly no amplification for all except my positive control :C.
Possible explanation is that I am having PCR inhibitor in my sample? What is the function of 6M salt in the protocol? How to get rid of salt contamination beside wash with 70% EtOH? Smearing in PCR product indicate presence of inhibitor?
I am lost. Please share with me if you have any thought. I will be truly appreciate for the help
Hi,
your DNA looks quite good and I do not see any problem in the protocol. 6M NaCl is added to precipitate digested proteins (additionally, I would recommend to incubate it for at least 30 mins on ice, to facilitate protein precipitation).
Based on your gel - it looks, there is qiute strong smear - that might be the clue. Sometimes it can be formed during PCR (if the overall conditions are far from optimal), but in that case, you should not get band in positive ctrl.
The other possibility is DNAse contamination - during salting out, there is no step, capable of 100% DNAse inactivation (proteinase K should do most of the work) - just the presence of EDTA stops DNAse from action during isolation. It might be, there is residual DNAse in your purified sample. DNA is not degraded, as the pellet has been washed - there are no divalent cations required for DNAse action. As soon as you add Mg (in a PCR master mix), DNAse can renature and degrade your template. Try to incubate your template in 1x PCR buffer (without polymerase,dNTPs - just buffer, MgCl2, water) at 37°C for an hour and check it on the gel. If degradation occurs, it is highly probable, it is DNAse-kill it with phenol/chloroform.
Good luck.
One more thing - if it is not DNAse, you can try to confirm/exclude the presence of PCR inhibitor. Just run your PCR with a mix of templates (your new DNA and your positive ctrl). If this reaction is ok, there is no inhibitor and the problem is in the DNA (can be overdryed, etc...). If there is no product, presence of inhibitor is likely - try to re-precipitate it with EtOH.
hi, I do agree with Martin Lenicek, that was a good explanation. Usually we do follow phenol-chloroform method for isolation. the second explanation by Martin Lenicek will be a better method to rule out.
It might help if you tell us what the dilutions are in relation to the lanes: to me that is not a series as some lanes give a stronger smear than others.
It would also help to know the %agarose and where the markers run: I see no evidence of PCR primers in any of the lanes, but it might be that they have run off the bottom of the gel. Also the DNA band in lane 1 seems remarkably monodisperse.
In my view there will be no active DNases in your prep, and even if there were, they would be completely inactivated at the initial denaturation step in the PCR.
There is no RNAse treatment in your extraction protocol, so you probably have a mixture of DNA and RNA, and your nanodrop readings will not reflect the DNA content.
And the PCR conditions, including for the +ve control (which you could add to your diluted DNA so ensure that you still get a band!
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