Hi, have you done a serial dilution with your DNA? If there are some inhibitors in your DNA, go down with your DNA concentration for the PCR (so you are diluting the inhibitors as well). Try if this will work. Good luck! Cheers, Nadine

So you should have enough DNA. I never worked with plant DNA, but you should exclude something strange - could you run the DNA just on a Gel electrophoresis (with and without maybe some restriction enzyme) to see if it is OK or, for example, heavily digested (even without restriction enzyme). In case the enzyme may not digest your new DNA and it looks otherwise intact, you may have a hint of something is even bothering the restriction enzyme (which also may affect PCR reaction).

But the simplest thing I could suggest: just precipitate your DNA again, or if that won't help, dialyse the DNA for 1-2 days in dialyse bags to remove any potential PCR enzyme inhibitors.

Hi, have you done a serial dilution with your DNA? If there are some inhibitors in your DNA, go down with your DNA concentration for the PCR (so you are diluting the inhibitors as well). Try if this will work. Good luck! Cheers, Nadine

It sounds like the Mature root samples contain molecules which act as PCR inhibitors. As Nadine mentioned, I would start with a serial dilution of your DNA. Also, you could add BSA to your PCR mix. (Even if your current master mix contains BSA, you could add some more). BSA has a tendency to "bind" inhibitors. You can add BSA to at least 0.5ug/uL with no problems.

Dear, Tania,

i also Agree with above suggestion. i also suggest what robert suggest. u are not included what is the DNA concentration u r using. For PCR it should be more than 1.8 (260/280) but if not , should not be difficult. I suggest root part should not contain phenolic compound so using PVP is not helpful , Please check.

now u can do 4 steps... (1) check the DNA ( RNase treated) and with out RNAse treated DNA on gel 0.8%.. (2) u take RNase treated DNA for PCR if it gives good quality. (3) Before PCR check ur primers with the BLAST to ensure ur primer is correct or not. (4) In PCR Different DNA concentration from 100ng to 500ng or more concentration u may use, and with high mg and high Tm as given in ur primer set in different set of reaction u can use.. hope this suggestion may favours u...

Dear all,

Thanks a lot for all your suggestions. I forgot to mention indeeed that I had already done a 1:10, 1:100 and 1:1000 dilution. And had no success. But maybe it is worth while to try that approach again adding the BSA to the mix as Mark suggested (thanks, I did not know that).

I will now check the DNA on agarose gel 0.8 % has Robert and Gopal suggested and let you know on the results.

Thanks once again for your prompt responses, which gave me new directions and, above all, hope of success.

I once had a mentor who worked at Agrigenetics (a biotechnology company that specializes in the genetic manipulation of plants). She told me that the best protocol for plant DNA extraction was actually phenol chloroform. A protocol for that can be found on the OpenWetWare Protocols page (http://openwetware.org/wiki/Phenol/chloroform_extraction). I have found this works for many applications, but still may not be robust enough for some applications. The OpenWetWare page also has a protocol specific for rapid isolation of genomic DNA from plant tissue that can be found at: http://openwetware.org/wiki/Rapid_plant_gDNA_extraction

As a disclaimer I have not one the rapid protocol myself and I only know what I have read about it. I hope this helps. Best of luck.

Zach

Dear Tania,

To find out if you have PCR-inhibitors in your sample you can spike some positive-control DNA to your sample and compare this to a pure positive-control DNA. If PCR inhibiton occurs you will get a weaker signal in your spiked sample.

Did you test Phenol-Chloroform DNA extraction? The extraction deals with toxic substances, but in the case of "dirty" samples this methode might work. Furthermore, I would suggest to test even more DNA extraction kits e.g. the Mobio "power soil DNA extraction kit" (I have good results with this kit when dealing with environmental samples that contain numerous inhibitors).

Thank you Zach and Silke. Indeeed, the CTAB protocol that I used had a phenol chlorofom step for cleaning up. Nonetheless, it migh make sense to do one extra DNA percipitation...

maybe you can try this protocol:

http://link.springer.com/article/10.1007%2FBF02823994

DNA extraction

Lyophilized carrot leaf, root, or inflorescence were utilized as a source of DNA.

To lyophilize samples, fresh tissue was wrapped in cheesecloth, frozen in liquid

nitrogen, and held at -80~ until transfer to a Virtis 12XL lyophilizer for 3-4 d.

Dried samples were then reduced to powder by grinding with a mortar and pestle

in liquid nitrogen. The powder was then collected on glassine paper and transferred

to 2 mL microcentrifuge tubes. Samples in this condition were held for up

to 13 months at room temperature. For comparison, fresh tissue was analyzed by

grinding with liquid nitrogen, transferring to a microcentrifuge tube, and DNA

was extracted immediately.

To approximately 200 IxL (~50 mg) of powder (visually estimated from

markings on the tubes), 800 IxL of sterile extraction buffer (50 mM pH 8 Tris,

0.7 M NaC1, 10 mM EDTA, 1% CTAB (hexadecyltrimethylammonium bromide),

and 0.1% [3-mercaptoethanol) were added. These samples were mixed and incubated

at 60~ for 45 to 60 min. An equal volume of chloroform:isoamylalcohol

(24:1) was added and mixed gently for a few min. Samples were centrifuged for

10 min at 8000 rpm, and 700 pL of the supernatant was transferred to a fresh

tube, 700 p.L of isopropanol was added and the contents of the tubes mixed gently

for a few min. The DNA was collected by centrifugation for 10 min at 8000 rpm

and the pellet was washed with 70% ethanol for 15 min without agitation. The

ethanol was decanted and the pellet was dried 6 to 8 min under vacuum or 10 to

15 min at 50~ The pellet was dissolved for 10 to 15 min at 60~ in 200 jIL lx

TE. Fresh tissue samples were prepared by the same procedure except 500 mg of

tissue was initially mixed with 80 tlL of 10x extraction buffer. Extracted DNA

was stored at 4~ for several days or at -20~ for long term storage.

Ok... it seems that, in spite of what nanodrop says, my DNA is degraded (or even almost absent, as nothing can be seen on the agarose gel; i.e, when nanodrop suggests 10ng/ul of DNA and I load 10 ul, I still do not see anything). Thanks.

What to do?

I reckon that the problem keeps on being on the extraction. Mature wild carrots look a bit woody and therefore, I have used liquid nitrogen followed by an homogeneizer to try to grinded it to powder before lysis. That seems insufficient. And I also do not have great amounts of material to work with (with luck, ca. 30 mg of root).

Phenol-chloroform extraction might worth a try, I guess. Something else?

Thank you. Once again...

30 mg of the tissue seems to be a lot for DNA isolation for PCR. I suggest you to set-up this PCR on regular carrot available on the market. Thus you will have the opportunity to use a leaves as an positive control without the possible root contaminants…

It seems that my initial suggestion of checking your DNA on an agarose gel was OK - Thank you for the feedback.

So the gel showed that your DNA was not OK - degraded or not existent.

Phenol-chloroform extraction should always work to get good quality DNA - you should use it.

Liquid nitrogen, powder grinding sounds perfect, too.

Dear Tania,

As Robert suggested (1) Perform powder grinding with liquid nitrogen (It is a difficult process), (2) isolate DNA by CTAB method (manually, do not use kit), (3) perfrom a phenol:chlorofom extraction and (4) precipitate DNA by ice-cold isopropanol.

Good luck

@Niroj: Why she shouldn't use a kit? Cheers, Nadine

You might try using the Biolase enzyme from Bioline with polymate additive. I have found this to be very useful for problematic amplifications involving plants.

I used to do genomic extractions on algal tissue and had similar problems. PCR was always difficult and we always ground the sample in liquid nitrogen and performed standard isolation techniques but always purified with phenol chloroform. As others have suggested, I would also recommend trying that, and you might even perform multiple extractions with the organics, sometimes a single extraction is not enough.

What is the source for your (+) control template?

If you are concerned about your DNA degrading over time, make sure you are using DNAse free reagents/tubes/tips etc. I know you probably are but if not, it's another possible source for contamination.

Best of luck.

What is the DNA amount (ng/ul) that you are recovering?

i have a suggestion, try to restrict (digest) your extracted DNA using one of the restriction enzymes (8 cutter or rare if you have) but usually i use BamHI, if it gave you a smear, it means that your DNA is good, if it gave you a sharp band, it means that you have a really good amounts of (enzyme) inhibitors.

another thing, what is your band size that you expect from this PCR reaction, if it is in a range

Thanks Niroj, but if it is ice-cold, after Phenol-CHloroform method, then a good amount of RNA, is going to participate in the elution too...

Tania,

It is likely that the roots of the more mature carrots are 'woody' and harder to break down - could you try a more aggressive treatment prior to accessing the Nucleic Acid ?

Another suggestion is to add in 'clean up'

I would like to add more BSA into either PCR or restriction digestion assays to alleviate the inhibition of contaminants. For example, using 10 x BSA to replace 1 x BSA standard often make me PCR successful when environmental DNA is used as a template.

I would also suggest diluting your sample to see if you then dilute out the inhibitors. In a completely unrelated set of samples, ie DNA from paraffin material, we do this even 1:10 to improve on PCR results. Else, there are also purification columns (eg QIAGEN) that further improve the DNA quality and thereby the potential to be amplified in PCR.

Good luck.

Ton

Its looks there is a problem in materials used and the glass wares contamination beside as Helen said breaking the cells of hard walls using LQ Nitrogen could be the solution to release the Nucleic Acid.

Try any of the two methods:

1. Use FTA cards or

2. try Extract-n-plant DNA isolation kit. from Sigma

Did you carry out electrophoresis of your sample's DNA and control? Try to see if there's any visible differencebetween your samples that could helpyou adjust the conditions (and check the accordance with the nanodrop readings).

What's the size of your amplicon? could a simple two step PCR reaction work in your case? good luck!

This is like with RNA, the yield and quality is satisfactory when you isolate DNA from young, small, dividing cells. If you have no DNA, try with still growing parts of the plant. Check the quality of DNA on agarose gel to compare the previous - good samples and new ones. After precipitation allow the precipitate to dissolve in water overnight or incubate at 60C.

I suggest you try an extraction buffer that contains spermidine and/or spermine. Numerous protocols are available. If it works good.

Mus

If you are getting a low 260/280 ratio, as Silke mentioned, I would recommend trying an organic extraction (phenol-chlorofrom) followed by dialysis against 0.1XTE or ddH2O to remove any of the organics which may be left behind. Your concentrations may decrease some, but your 260/280 should improve

I suggest to use the PlantDNeasy kit (Qiagen). This kit every times works for all taxa.

to avoid or reduce degrading of your DNA you may include a step with Proteinase K

Dear Olga Amaral,

I did check and indeed, as I expected, the DNA in the samples is more degraded than in the control. But there is still DNA in it, so it should work somehow. Why do you think a two step PCR would work? I am not familiar with it…

Thanks.

Tânia

Dear all,

Here is a (bad) picture of the extractions in agarose gel. As you can see, he DNA is degraded, but I have the feeling it should work as there is still DNA (as far as I can perceive). This extractions were made following some of the suggestions already given in this forum, and for those I thank you.

Tips on how to put te PCR working? My amplicon is about 1000 bp and with the control it amplifies with a standard reaction of 35 cycles and at a t(a) of 55 C.

Thank you once again,

Tânia

Hi, the picture should show us the DNA after the extraction (not after PCR), right?! Cheers, Nadine

yes, right! Sorry for the bad quality and the absence of lambda for checking... but we run out of it in the lab. oops!

And why do you think your DNA is degraded?

Hi Nadine, because I see a band and a smeer underneath and I was told that that means some degradation of the DNA. I have to confess that I seldom have checked DNA integrity on agarose before (I am trusting what I was told, no know-how).

But if that is not the case, then I even have less cues on why the PCR does not work! While it does for the positive...

Thanks.

Hi Tania, have a look here (also the DNA in my tested samples often looked like this and even AFLPs worked!): http://www.google.de/imgres?um=1&client=firefox-a&sa=N&rls=org.mozilla:de:official&hl=de&biw=1360&bih=622&tbm=isch&tbnid=H13DOr_w52aF8M:&imgrefurl=http://www.mobio.com/soil-dna-isolation/powermax-soil-dna-isolation-kit.html&docid=mu6vOksGExhsyM&imgurl=http://www.mobio.com/images/custom/image/12988gel1.jpg&w=288&h=251&ei=R5wcUtSXCe334QSczYG4CA&zoom=1&iact=hc&vpx=545&vpy=317&dur=380&hovh=199&hovw=230&tx=89&ty=115&page=4&tbnh=152&tbnw=191&start=82&ndsp=29&ved=1t:429,r:92,s:0,i:364

It's right that people say a smear means degraded DNA. But you can see the smear very often after DNA extraction (maybe it depends on material, contaminants, extraction protocal/kits,...). Maybe your template is difficult to amplify (older material)?

How high was the DNA concentration you used?

Cheers, Nadine

Hi Nadine,

I will certainly have a look at the kit that you suggested. The images seem convincing! ;-) Do you have experience with the kit for non-soil material?

Indeed my actual template is older than the one used as control. The extraction procedure was the same, but this new samples have been collected, posted and only then put on -80C for later extraction. Time ellapsed from collection to extraction varies from 2 weeks to 1 month.

For the PCR, the control that I am using is at ca. 10ng/ul. I have diluted the extractions (the same as the ones on the picture) to around the same concentration (given by nanodrop quantification), which corresponds to the traditional 1:10 dilution. I have also tried the PCR without dilution, actually, and in that case none has worked (also the undiluted control) except the control.

Cheers!

And I cannot thank you enough (and all the others in the forum) for the help and the oportunity to share my troubles.

Hi Tania,

I have a little bit of insight on the gel of your DNA extraction. I was unable to open the image from my computer, but based on your description it sounds like you are seeing a smear. Smears are actually very common after the isolation of genomic DNA. Basically this means the DNA has been nicked in various locations, but this is normal. The key is not all of the nicks are within the DNA sequence that you want to amplify (at least statistically). As a result the DNA is still good for PCR. I have done PCR on smeared genomic DNA isolations on many occasions, and the results are no different from the rare occasions when I have a smooth heavy band of genomic DNA to work with.

The first thing I would recommend doing is to check the absorbance of your DNA for quantification (using Beer's Law) using a Nanodrop or a spectrophotometer. Also check the absorbance at wavelengths 260 and 280 nm. Divide the 260 nm wavelength absorption by the 280 wavelength absorption. If you have a number that is greater than or equal to 1.8 your DNA is clean and should produce a very good PCR. With that being said I have done PCR on samples with 260/280 ratios as low as 1.3 with success. (And most of my PCR products are 2 KB and above).

Also note that the polymerase that you use may also make a huge difference. For instance some polymerases such as Phire polymerase are very able to amplify when the DNA is not as clean. Also if your PCR doesnt work it could be because of the size of your amplicon.

Hope this helps.

Cheers,

Zach

I am about to give up. With the new extractions, nanodrop measurements are between 45 ng/ul and 60 ng/ul and the 260/280 ratio ranges between 2.1 and (the lowest) is 1.6. I have tryed Phusion and goTaq. I have lowered the t(a) hoping that something would happen. I am off home now.

Cheers to all.

I think I can help. If you are comfortable sharing the sequence you are trying to amplify I can design the primers for you. If you'd like you can send me an email at [email protected], and I will see what we can do. I have been in your boat before, and I have made almost every mistake in the book (so I can steer you away from them).

1000 bp is perhaps just too long for bad DNA; I didn't see which method you recently used to get your DNA, any kit, which may not remove some bothering proteins? - Or did you use any old-fashioned (bullet-proof) phenol-chloroform extraction? with some proteinase K digestion? is there any possibility that you accidentally included some DNase; I don't remember, if one uses "DNas-free" RNase anywhere, but maybe you start with complete new buffers everywhere. Good luck.

Just my old anecdote: I once had all of a sudden no good Southern-blots anymore, when working in a rather strange lab at the Karlsruhe nuclear Research Center, where a student had stolen a genomic Library out of my fridge to base her PhD on. I repeated my Southerns-blots until a colleague told me his (bad) experience from another "competitive" lab with people on the same project often sabotaging each other. So it turned out, my previously working "neutral" transfer buffer was extremely alkalic, we excluded the theoretical possibility that we mixed up NaOH and neutral buffer - but we did not accuse our poor competitor - it could have been a strange accident or everyone to disturb anyones success in a highly sabotaging lab for many years. So this won't be the situation anywhere else, but you should confirm you have no pH 10 in your DNA solutions. NaOH pellets falling in your solutions may typically denature your DNA.

agree with Nadine A. Gund

Dear Tânia Nobre,

I kindly suggest trying QIAGEN DNeasy Plant Mini Kit if it is possible for you. After Wash step, repeat Eluting for two times by aspiration the passing solution after centrifugation and re-dropping it on the filter column, it will help you to increase the yield of your sample.

I hope it could be helpful.