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CTAB of Fungi

CTAB DNA Extraction Electrophoresis Fungi

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23 replies to this topic

#16 bob1

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Posted 21 August 2012 - 12:45 PM

The NH4oAc is the salt component of the precipitation, similar to adding NaCl or NaoAc. You need a certain amount of salt to get efficiently precipitate nucleic acids, these are often provided by the lysis buffer(s) but can also be added at the end. The amount of salt depends on the compound for some reason that escapes me at the moment.

#17 Axolotl9250

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Posted 24 August 2012 - 02:21 PM

Just tried a PCR amplification, no luck and no amplicons, when I consider PCR it's probably worth a new thread, but my purity values for my 5 samples I'll list here, if they're not pure enough I'll need to do them again. I'm not that experienced so I don't know what level of purity I can get away with. I know 1.8 is good A260/A280 and 2.0 is good A260/A230 however.

A260/A280 A260/A230 DNA Conc.
Sample 1 = 1.760 1.477 147 ug/ml
Sample 2 = 1.848 1.410 974 ug/ml
Sample 3 = 1.362 1.219 64 ug/ml
Sample 4 = 1.594 1.320 80.5 ug/ml
Sample 5 = 1.688 1.093 153 ug/ml

I've also done the protocol from post #13 but with the 50ul of 7.5M ammonium acetate and 2 vol. of ethanol. I noted a greater increase in DNA yield according to spec. Which is interesting given sources that say DNA is less soluble in Isopropanol so precipitation of larger species and smaller concentrations (although the same source also states ethanol is better for small volumes and small DNA concentrations). Does anyone have any advice as to the concentration of Ammonium Acetate for Ethanol precipitation? I've seen 0.1M or 2M or 50ul of 7.5M, and even 1.5M and then ethanol precipitation, followed by 200mM and another ethanol precipitation (no wash afterwards). Or is Isopropanol really best? - I seem to see conflicting opinions in the literature or on sites.


I think I need to consider ways to improve on those purity values, but I already have a phenol component to get rid of protein and I thought CTAB handled polysaccharides fairly well. Tomorrow I'm using 50mg of material and not 100mg, and I think maybe a smaller pipette to prevent possible phenol contamination, I noticed even if I'm careful with a p200 the boundary get's a little disturbed, I could take a conservative 200ul of the supernatant rather than my 400ul I manage with the p200, to ensure boundary layer is not disturbed. Use of a step where the supernatant is incubated at 4 degrees for 15 mins in 2M NaCl and 4% PEG is used to increase yield and 2M of NaCl is also said to help with polysaccharides, I don't know if anyone else knows of why this is or has even done it. The same paper also uses a wash solution of 15mM Ammonium Acetate in 75% Ethanol.

Is there anything I can do already to my DNA so I don't have to go through everything all over again. I'm thinking of also testing for DNAses by incubating at 37 degrees overnight.

As a final consideration - 1ul of my DNA samples are added to a working volume of 25ul for my PCR mix I'm wondering if, impurities considered and the concentrations of my DNA, if it's just too much.

As always your hints and advice are greatly appreciated, I've been reading a lot but it doesn't make up for being a 1st molecular project newbie.

Edited by Axolotl9250, 24 August 2012 - 02:31 PM.


#18 Adrian K

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Posted 26 August 2012 - 07:26 AM

Just a little piece of thought. Did your template work in other primers set? This is because I think there is a very high possibility that your primers are not working rather than you have a bad template. Based on my memory, I used to have such quality template to do my PCR and I still got my amplified amplicons.

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#19 phage434

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Posted 26 August 2012 - 09:05 AM

The ethanol will not precipitate DNA without the presence of salt. The ammonium acetate is the salt in this situation. Certainly the resuspension and reprecipitation will remove contaminants from the first preciptation, but it will not remove the salt (ammonium acetate), which is the purpose of the 70% ethanol wash in the standard protocol. The vacuum may remove the ammonium acetate, given enough time, since its decomposition products are volatile. The DNA will be very hard to resuspend after this, since it will be so dry.

#20 Axolotl9250

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Posted 27 August 2012 - 12:04 PM

I've tried another PCR reaction and it has worked this time, albeit I need to re-run it because I got some smear in my negative control. However the expected RAMS bands predicted to appear based on a previous student's experience did appear. I'm not entirely sure why the first reaction did not work, perhaps DNase, but if it were in my reagents then I would have expected the run that worked to fail as well. I have noticed I have a tendency to over-dry my DNA, but after a day or so of heating for a few minutes and then mixing sorts this out. My next goal is to try and vary the component amounts to reduce some of the noise and get an optimal result.

#21 bob1

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Posted 27 August 2012 - 02:15 PM

Sometimes with these sorts of reactions, especially with plants and fungi, they contain a lot of inhibitors that may cause the reaction to not work if the DNA prep is used directly in the PCR, however they may well work if the DNA prep is diluted, often 1:500-1:1000 seems to be a good range.

#22 Axolotl9250

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Posted 30 August 2012 - 07:38 AM

I did my PCR and ended up with a single bright band (even in NTC), very small - near the level of the last band of my 100bp ladder. Am I looking at contamination or primer dimers? Absolutely nothing else was amplified and there are no smears. Just this one band in every lane. If so then I think I'll have to convince the two I'm working with the primers are no good and we need to try something else. I did everything in a flow hood I cleaned with a DNexitus plus solution, pipette tips straight from the autoclave too, sadly my pipettes are not autoclavable.

#23 phage434

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Posted 30 August 2012 - 07:50 AM

I suspect you have primer-dimers. I never autoclave tubes. In my opinion, dirty autoclaves are the last place you want to put something that is supposed to be DNA free. Out of the box, they are essentially DNA free, especially if you are not working with human DNA.

#24 Axolotl9250

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Posted 30 August 2012 - 02:47 PM

This is awfully strange, do primer dimers often mean no desired amplification? I would have thought I'd get them with some amplification. I did the same thing the previous PCR attempt and got desired amplicons and a bit of smearing so this time I was using different template amounts of 20ng 50ng and 100ng to compare them. The only thing I think I did differently was I added my primers to the buffer and dNTPs before template (on ice). The last time I dotted my primers and taq onto the side of the tube (I still did it with taq) so as upon heating they fall into the buffer, template & dNTPs and start the reaction. People have described making master mixes with primers in though so I didn't think it mattered much.





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