Hi, I'm extracting bacterial DNA from CTAB/NaCl method. Starting material is either colonies or culture grown in LB broth. I follow the standard method, heat kill, lysozyme+TE 37C 3hrs, then Proteinase K 65C 10 mins, followed by NaCl, CTAB, Chloroform:Isoamyl alcohol for phase separation, then Isopropanol overnight, followed by ethanol washing. DNA is dissolved in 0.1x TE.
My problem is, some of the DNA work and some don't for 16S PCR. when the 0.7% gel ran, many looked as a smear, sheered, while some stuck in the well. Conc. is measured using quantifluor, got around 100ng/ul. Of this, 5ul was added (500ng) for PCR. Also, some have good bands of gen DNA, PCR doesn't work.
My conclusion is there are inhibitants such as proteins or salts. Does anyone know how to get rid of them? I do phase separation & ethanol washing twice even. No luck.
Thanks!
Hi, from what I see the extraction it's not the problem since you get 100ng/ul of DNA, so I agree with you there must be some pcr inhibitors, usually after the DNA extraction I go and do a DNA purification with Qiagen magnetic beads, you can lose a little bit of DNA but it won't be that much. I've tried with ethanol washing also but it doesn't seem to work very well.
Anything further questions just ask!
Nuno
I think that you are using too much DNA. if the dna is pure then 25-50 ng is fine but using too much sample increases the risk of inhibitors being present, I would try adding a little more magnesium and a pcr of dilutions of your dna. It may be that you have other substances which look like dna present in your sample in which case you do not have as much dna as you think and re purifying as Nuno says should help. If you think that you have inhibitors present try a pcr with a dna that works and also include a samp,e of the dna that works with an equal amount of dna that does not work added. If there are inhibitors then the added extra dna will kill the working pcr reaction but the simplest first try is using less sample
Hi Nuno, Thank u for the reply. I appreciate your suggestion, but could you please suggest something alternative for that? We're running out of time for this, so can't order now.
Hi Paul, Thank you. We've tried with low amount of starting material. Again no result. :(
phenol/chloroform is still a very good purification method and worth a try if you have phenol around the lab
Dear Anur
I agree with Nuno: I think your PCR is being inhibited but I think the problem is salt based on a massively excess incubation period with isopropanol, which will bring down salts; Let me expand:
- To be specific, isopropanol precipitation is designed to be performed for about 15 min at room temp; not overnight at room temp or not for 15 min at -20C, unlike ethanol precipitation. If you incubate with isopropanol at -20C for 15 min or over night at room temp; or worse still over night @ -20C you will bring down a massive amount of salt and other impurities that, like Nuno said, will inhibit your PCR. In addition, the large salt load could in part account for the smearing of DNA on your gel
- However, I think the other reason you might be observing smearing is that your extraction method is too severe, which will definitely result in loss of DNA but also degradation of that DNA; Its as if you have combined about 3 different methods of DNA extraction all of which I have used extensively in the past
- In particular, for bacteria, if you add lysozyme to break open the cell wall - a very good idea, particularly for midi or maxi preps -then with prot K digestion you can probably dispense with the CTAB step: Indeed, by adding in CTAB and Nacl and then precipitating over night with isopropanol, it is likely you will have residual CTAB and NacL in your final purified pellet, which will definitely cause smearing and definitely inhibit PCR
This is how I think you should proceed
Dear Laurence,
Thank you for the suggestion. That actually makes sense. Thanks again!
You could try lowering the concentration of EDTA in your TE buffer - EDTA chelates Mg2+ which can affect PCR. A storage solution of TE(0.1) may be worth a try; use 0.1mM EDTA rather than the 1mM in a standard TE solution.
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