CTAB DNA extraction - Science method

Jaber Amin

The extraction of DNA from the thoracic region of honey bees using the CTAB (Cetyl Trimethylammonium Bromide) method can sometimes be challenging due to the presence of various compounds that can interfere with the DNA extraction and downstream applications like PCR.

Here are some potential reasons for the observed issues and suggestions to improve the DNA extraction and subsequent PCR results:

1. Phenolic compounds:

- Honey bees, especially the thoracic region, can contain high levels of phenolic compounds, such as flavonoids, which can co-precipitate with the DNA and inhibit enzymatic reactions like PCR.

- To address this, you can try adding a higher concentration of PVP (Polyvinylpyrrolidone) or β-mercaptoethanol to the CTAB extraction buffer to help remove these phenolic compounds.

2. Polysaccharides:

- Honey bees may also contain high levels of polysaccharides, which can co-precipitate with the DNA and lead to the observed sticky or indistinct PCR results.

- To mitigate this issue, you can try increasing the salt concentration (e.g., NaCl) in the CTAB extraction buffer or including an additional precipitation step with isopropanol or ethanol to better separate the DNA from the polysaccharides.

3. Protein contamination:

- The thoracic region of honey bees may contain high levels of proteins, which can also interfere with the DNA extraction and PCR.

- To address this, you can try increasing the proteinase K incubation time during the CTAB extraction or including an additional phenol-chloroform extraction step to remove the protein contaminants.

4. RNase treatment:

- The presence of RNA in the extracted DNA samples can also contribute to the observed issues. Consider including an RNase treatment step during the DNA extraction process to remove any RNA contaminants.

5. DNA quantification and dilution:

- Ensure that you are using the appropriate amount of DNA template in your PCR reactions. Try diluting the extracted DNA samples to find the optimal DNA concentration that works best for your PCR assay.

6. PCR optimization:

- Review and optimize your PCR conditions, such as primer design, annealing temperature, and cycling parameters, to improve the specificity and sensitivity of the PCR.

7. DNA purification:

- Consider using a commercial DNA purification kit after the CTAB extraction to further clean up the DNA and remove any residual contaminants that may be interfering with the PCR.

By addressing these potential issues, you may be able to improve the quality and purity of the extracted DNA, leading to more reliable and distinct PCR results.

Hope it helps; partial credit AI

Incubate the sample with ctAb at 37°C for 6-8 hours with minimal agitation. Alternatively, incubate with agitation on the shaker at 55°C for 30 minutes to remove fats and lipids. Then continue to use your regular isolation method.

the dna is partially degraded but the fragments amplify well as proved by your working pcr. I do not see a no template control (NTC) where you amplify water instead of dna. It should be negative of course but when we see a smear of high molecular weight amplification then it looks like contamination of the reaction with pcr product. Run a NTC sample and if this produces a smear of amplified material then you will need to get rid of the contamination but first run a NTC as well as a sample then take it from there

Agreed with Nouh Saad

Re amplification must be treated with care. In early stage pcr every 10 cycles is 1000 times more product so if you use too much template or too many cycles the amplimer will self anneal and form smears of lonmers. I would use 1ul of first round product...dilute it into 100ul and use 1ul of this dilution. Set up 6 tubes of pcr and set for 24 cycles and remove a tube at 12,14,16.....24 cycles and see which cycle number gives good clean amplification.

A cleaner way is to design an internal primer set just inside the first pcr primers. They can be quite poor as primers because the template is almost all the correct amplimer.. Run 20 cycles of a 1% dilution of the first round product ( 1ul of 1/100 dilution) and it should give a clean product. There is no need to clean up the first round product between amplifications

After adding isopropanol store this sample at -20 for overnight. This will also enhance your DNA concentration.

Many Thanks

PVP is basically a polymer, which will only mention as %. So its better to go with dilution. In my experience, CTAB-buffer with 2% PVP works well for the Isolation of DNA. Incubate your CTAB with PVP for 15 mins at 65 C and then vertex.

Dear Tayebe

I am not sure I have understood what are differences between the lanes (and the percentage of agarose too, and DNA ladder size too), but I would say that right two lanes on the bottom part look like a good genomic DNA. Other samples in my mind show degraded DNA. I also would say that the gel is heavily overloaded, so I would load less material. This can help to see if the degradation is full or partial. BTW, the physical forces applied to the samples can cause DNA degradation - another reason to use as less time as possible for homogenization in lysis buffer. Also, additionally to integrity of the DNA, you can check the purity of DNA by NanoDrop. Since you are trying to find the best procedure, I would aggregate all this information before making decision and accepting it as SOP. But good sign - your homogenization generally works - you do get tissue disruption.

Which method did to use for separation if a third component (solvent) was not used?

The Ethanol-water binary system form an azeotrope unless the pressure is below 11.5 kPa

Hi Rahul,

I tried with Qiagen DNeasy Plant mini Kit with modification, u can use filter paper method or plankton from net for extraction, but freeze throwing at Liquid nitrogen, and homogenization using bead beating is essential and after that incubation at 3-6hr at 55C

please refer: Nowinski et al.,2019(Nature -Scientific data)

Very unlikely. DNA and RNA share many of their physical chemical properties, so they cannot be distinguished without specific enzymatic activity. RNA is more liable to heating, though, but required thermal treatment will not most probably leave DNA intact.

Results so far: worst extracts with QIAGEN DNeasy, CTAB (McFadden, Coffroth protocols) worked well, best result: salting out with modifications. As expected, preservation methods and sample age majorly influence the outcome.

Hello María José,

do you perform a PCR on the extracted DNA and send the PCR product? If yes, the salt contamination is more likely to arise from the PCR mix. I can recommend a PCR clean up step either by column or gel extraction.

If this is not the case I would recommend an additional 70% ethanol wash as I assume you extract with isopropanol? Salt and DNA precipitate, but not in ethanol.

10 ng may not be enough to visualize on the gel

Nirmal

you can have a look on this article, you will find useful tips to avoid DNA degradation:

When you isolate genomic DNA you will also have plasmid DNA included, and in fact likely to be over represented in the mixture. It is possible to purify the plasmid away from the gDNA based on supercoiling properties, but it is generally not easy to purify the gDNA away from the plasmid. You will have both.

Hello, by benzylchloride method you can extract both plant and fungi DNA by same procedure and same reaction mixture and you will receive both DNAs mixture. If you want to isolate only fungi DNA the better way is to isolate fungi culture from your material as pure culture and then perform DNA isolation.

Dikeos Mario Soumpasis

I wanna use hyaluronic acid-based hydrogel particles for gene expression on microscale. I know, hyaluronic acid is not the best candidate as a polyanion. There are two reasons why I am aiming for hyaluronic acid based platform. The Gels need to be porous enough to allow for diffusion of the ribosomes throughout the particles. Second, the HA is further functionalized with binding motives and therefore HA needs to be quantitatively functionalized (possible due to the carboxyl group of HA). So switching to chitosan or agarose is not really an option.

Actually i have found in: "Protocols for Nucleic Acid Analysis by Nonradioactive Probes" ed Elena Hilario protocol for isolation from spores that use PVP 360:

Put 10 pretreated glass beads into an microcentrifuge tube containing the spore sample.

2. Add 0.2% β-mercaptoethanol and 1% PVP-360 to the CTAB buffer. Add 200 μL hot (65°C) CTAB buffer mixture to each tube (see Note 22)

3.Vortex for 1 min at high speed.

4. Incubate at 65°C for 30 min.

5. Add 200 μL chloroform/isoamyl alcohol (24:1).

6. Centrifuge at 14,000g for 15 min.

7. Precipitate with 2 volumes of 100% ethanol and leave sample at –20°C for at least 3 h. Repeat step 6.

8. Wash with 2 volumes of 70% ethanol, and repeat step 6. Discard supernatant.

9. Dry in a vacuum centrifuge.

10. Add 10 μL TE buffer. Store at 4 or –20°C.

Hi khem

Your pellet may be completely over dried. So many of them face a similar problem (sometimes I had a problem to dissolve the pellet) and sometimes heating the solution to almost boiling T may be useful for a short period of time.

But that may degrade your DNA.

Good luck

In order to purify DNA, insoluble particulates are removed through centrifugation while soluble proteins and other material are separated through mixing with chloroform and centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is then resuspended and stored in TE buffer or sterile distilled water. This method has been shown to give intact genomic DNA from plant tissue. To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with ethidium bromide, and visualised under UV light

Dear Kamesh , my suggestion is: use a method based on chelex. You can use with just one specimen.

Improving the quality of DNA means to remove other contaminants from it.  Generally treating DNA with RNAses is recommended to yield high-quality DNA.

Again responding after many years.

To get DNA from the cell we need to break the cells so that the contents of the cell come out including NUCLEUS where the DNA is present. To break or lysis the cell wall including the wall of the nucleus we have to create the osmotic pressure so that the wall bursts and cell contents release by the action of proteolytic enzymes along with sucrose that present in the extraction buffers. Hope it is clear why we add sucrose in buffers.

1. A certain amount of cells equivalent to 1 OD₇₃₀ units of culture were harvested by centrifuging at 12, 000rpm for 1 min;

2. Adding silica sand.

3. Then, 100ul saturated phenol (saturated phenol is in the lower layer, upper level is protection liquid) and 100ul Chloroform: isoamyl alcohol (v/v 24:1) were added. And cells were disrupted with silica sand and those organic regents by vigorous vortex (5 min);

4. After centrifugation at 12, 000rpm for 5min, you can collect and transfer the supernatant into a new tube . Then you can add Chloroform: isoamyl alcohol (v/v 24:1), whose volume was similar to the supernatant. After mixing with chloroform and isoamyl alcohol thoroughly and centrifugation, the supernatant was collected.

5. After the addition of 1/5V(Volume) NaCl and 2~3V ethanol (100%) , you can mix them thoroughly and then set them at -20℃ for 30min at least.

6. After centrifugation at 12, 000rpm for 10min, you can remove the supernatant. Then you can add 500ul ethanol (70%) to wash the DNA.

7. After centrifugation at 12, 000rpm for 5min, you can remove the supernatant. Then the precipitates were collected and dried at room temperature for 3~5min.

8. You can add 30~50ul ddH2O or TE to dissolve the DNA.

Thaks a lot.

LiCl  increase yield and stability of the RNA preparation for tissue samples especially plant  species and improve cDNA synthesis.

You can add more than once extraction step with chloroform too. 

How old is the chloroform that you have? How long are you centrifuging the sample for following chloroform addition?How much did you add chloroform?all are the key factors that influence chloroform extraction step.

following papers may help you

I would start by preparing a new batch of CTAB. The recommended shelf life is 2 weeks. I had a similar situation and realized I received many attempts later that it was the RNAse A. I moved the RNAse step after phenol chloroform isoamyl alcohol extraction and incubated at 37C for 30 minutes, followed by a chlororm isoamyl alcohol extraction. You can also make sure you are grinding into a fine powder before adding heated CTAB.    Increase your incubation time if needed, after adding CTAB.

You can also increase your salting out phase. Try leaving at -20C overnight or you can try letting your pellet dissolve overnight. Are you getting a pellet? If so, what is the consistency?

Sodium bicarbonate is used as anticaking agent or it also helps in color retention because when coffee absorb moisture it color changes from brown to dark brown or vlack

Thank you very much Ivan

I think this will help you

 Dear Rene van Wezel,

Thanks a lot for the information and articles. I would also like to thank Bhaskar Gouda, Jetty Ramadevi, and Shreeti Pradhan for your valuable comments

Thank u all

Use Quick-DNA™ Plant/Seed Miniprep Kit, it helps as we have isolated genomic DNA of chickpea seeds.

Thank you all

As any ratio your 260/230 is a result of two value. Either 260 (DNA is low) or 230 is high. It is therefore interesting to look at the value independently, is your 260 value good (compared with nice DNA which you can borrow from a fellow lab member or is your 230 high compared with the DNA from your fellow lab member. I think before troubleshooting your protocol you first need to know which of the two problem you are facing, are you having a problem recovering your DNA (low 260) or extracting to much of 230 absorbing compunds (high 230). Get this info first, then troubleshoot.

What kind of 'bad quality' did your isolated DNA have?

Did you grind the fresh tissue well enough? Becoming powder in the liquid N2?

Were your kit or CTAB solution old?

Ethanol has higher dielectric constant than water and will thus "soak-out" the water from nucleic acid and leave it dehydrated. Now positively charged ions in your solution can access nucleic acid and form salts.Without Ethanol (or propanol) positively charged ions don't stand a chance at accessing phosphate groups in DNA because they're surrounded by water molecules.

You may be need to change the pH of solution  or increase the concentration of ammonium acetate. I wish this helpful. 

Hi Sakinah,

Do you need the amount of DNA extracted to be replicable, ie. be fairly consistent across samples?

If not, CTAB might work for you. We've had it work on a range of difficult samples (salt water, not peat though) and it's been consistently giving us better results than the PowerSoil kit, as well as a few others tested. However while the DNA concentration is higher than the kits, it's quite variable. Would not recommend for qPCR but for PCR and ID, it's great.

Let me know if this interests you and I'll dig up the protocol.

Thanks for your advice Artur Burzynsky

Hi René

I had same question months/year ago but was hard to find information. What I found was that the critical micelle concentration (CMC)  and micelle microviscosity for these two cationic surfactants, have been studied through Electron Spin Resonance technique and the main difference seems that self-aggregation behaviour seems to be more marked and cooperative for CTAB than for DTAB thanks to the higher number of carbon atoms in CTAB tail. It occurs around 1 mM for CTAB and 14 mM for DTAB. However this does not reply to your question. Mixture of them is recommended for dirty samples apparently.

As their carbon chains are different (12 and 16), I am wondering as their charge is positive if their zeta potential is also different and if the target proteins they bind could be different, therefore having a mixture could be the best indeed to get cleaner DNA. In the group, we use CTAB and it worked quite well however but working with algae…

If you want to extract the DNA of the microrganisms living in the plant, you may simply use typical DNA extraction kit to extract all the DNA first. 

After doing NGS, you may use filter to filter out all the reads from plant genome. 

Dear Nilay

Your DNA samples are carrying too much protein contamination. It seems to me handling problem. Extraction protocol looks standard but there are many things you are getting even after standard procedures, for example, you perform Phenol treatment for protein removal and RNAse treatment for RNA removal, but you still can see RNA at the bottom of your samples. The DNA samples also have degradation as smears can be seen in most of the samples.

Suggestion:

1. Check your pippetting, take care during picking supernatant that you should carefully pick the liquid only from upper layer.

2. After grinding leaf tissues in the pestle and mortor, transfer the powder into 2 ml tube, where you can add prepared (2X CTAB + 1% pvp + 0.2% b merceptoethanol). Add PVP and merceptoethanol into CTAB right before use.

3. Try precipitating DNA with isopropyl alcohol.

4. Did you perform RNAse removal after RNAse treatment.

5. Try using Phenol:Chloform:isoamly alcohol (24.1.1)

I really think the problem is only in handling (handling error), otherwise your protocol seems fine.

Best of luck

Hi, Nibras

I will keep that in mind.. Thank you..

Ok, thanks for the advice

There is also a very good article regarding DEPC and RNA from ThermoFisher Scientific company. I recommend you to read it. The title of the article is:

possibly the colour is caused by humins  which often purify with dna. You could try ethanol precipitation ( with glycogen added if you do not have very much dna as a co precipitant then do an OD260/280 to ascertain dna purity Dusan makes a good point that when you do have interfering substances sometimes using less samples allows a PCR to work rather than using more and having more interference. Having said that any substance that absorbs at 260 will make you think that you have more dna than you actuallu do have

grapevine leaf

Most likely it is still denatured proteins/excess salt etc. It is often required to do several rounds of phenol-chloroform extraction to remove all of the proteins. You need to repeat phenol-chloroform extraction until no precipitate is visible and then perform chloroform extraction step. 

Hi, Sharmeen Rahman,

I am extracting DNA from shrimp frozen tissue that apply to GBS method. I used kit (Invitrogen), phenol: chloroform:isoamyl alcohol (25:24:1) but they appear to be a smear. Can you help me?

i think is problem of your DNA, perhaps after one week it gets degraded because of some contamination that remains in your the DNA. I suggest to check the quality of your DNA after storage and to adjust DNA extraction conditions if you find that it degrades after a week. Also consider this situation of multiple bands... how big is the amplicon that your are expecting? It might be also due tu unspecific DNA amplifications?

You can use another conventional protocol, there are several procedures to obtain good results or Wizard genomic purification kit Promega (only for high yields), but in my experience DNeasy is an excelent kit. May be, you could try to freezing the mycelium (verify the amount of mycelia) before extraction techniques. Celular lysis is fundamental step previous to DNA extraction. I recomended to use lyophilized material. Good luck :)

Dear Oadi,

phenol is used for the separation DNA and proteins during extraction process. It allows to precipitate coagulated proteins in the interface between aqueous and phenol layers.

Best regards,

Maxim

DNA is washed with 70% ethanol to remove some (or ideally all) of the salt from the pellet. If water was used as the wash then DNA would dissolve again and if 100% ethanol was used the salt would not wash off because sodium salts are poorly soluble in ethanol.

because precipitation in 100% ethanol cause removal of all water molecule from DNA and Complete Dehydration,which make them not soluble, So we give 70% wash to let it retain some water molecule when make it soluble.

Hi there,

I have run into a similar problem and am quite interested in the column clean up procedures you used. Ales, I have found the types columns you described. They are quite dear and only purify up to 100 microlitres of sample. Steve, which columns did you use? Did it resole your issues with the qPCR? Because that is my end goal too.

Thanks!

Hi all, thank you very much for your advice. Just to update....We checked the pH of the CTAB and that was fine. There were already two ethanol washes in the protocol. The RNA contamination is important in this case as the samples are to be used for BS-sequencing.

As a trial, we ran the extraction process without any RNase treatment and found that c. 50% of the sample was RNA. So the RNase treatment is working to some extent as it reduces the percentage of RNA in the sample to 15-20%. What seems to have been going on here is that by only using one type of RNase the enzyme is not chopping up the RNA into small enough fragments to be soluble in the ethanol and as such it is still precipitated out along with the gDNA.

I was advised to try RNase T1 or a Riboshredder as opposed to RNase A.

Thanks again for your help!

I agree with Al- Amin suggestions.  You may wish to look at the Vilgalys CTAB protocol available at http://www.umich.edu/~mycology/protocols.html.  I have had similar "protein smears" on gels which I believe was due to my lack of "carefully" (see Al-Amin note and Vilgalys instructions) removing the upper layer in the Chloroform:isoamyl alcohol extraction.  

Some fish eggs (salmonid ones) have a very strong "capsule" (zona radiata, zona radiata externa and vitelline membrane) which are impermeable to most substances (the salmonids' vitelline membrane is even known to be impermeable to water and the zona radiata externa prevents the whole capsule to be digested by external proteolytic treatments). Accordingly, preservative substances won't be efficient if you don't preliminarily break the capsule.

Yes Javier, you can easily treat an aliquot of the DNA preparations with good quality DNase free RNase A. After RNase A treatment, you can either use the DNA preps directly for PCR or other applications or else can re purify the extracts with phenol-chloroform extraction-ethanol precipitation to remove RNase A completely.

Best wishes

SD

Principles of CTAB method: lysis, extraction and precipitation

Plant cells can be lysed with the ionic detergent cetyltrimethyl ammonium bromide (CTAB), which forms an insoluble complex with nucleic acids in hypotonic medium. Thus, polysaccharides, phenolic compounds and other contaminants remain in the supernatant and can be washed. The DNA complex is solubilized by increasing the salt concentration and precipitated with ethanol or isopropanol. Lysis of the cell membrane, extraction of genomic DNA and its precipitation: In this section the principles of the three main stages are described.

 In the extraction step, the complexes formed by the CTAB nucleic acids and polysaccharides, phenolic compounds, proteins and other cell lysates dissolved in the aqueous solution are separated. It is especially important to remove polysaccharides and phenolic compounds, they may inhibit many enzyme reactions. Under low salt concentrations (<0.5 M NaCl), contaminants nucleic acid complexes do not precipitate and can be removed by extracting the aqueous solution with chloroform. The chloroform denatures proteins and facilitates the separation of the aqueous and organic phases. The aqueous phase is usually the top phase. However, if this phase is dense due to salt concentration (> 0.5 M), will form the lower phase. Also, if the pH of the aqueous solution is not properly balanced (pH 7.8 to 8.0), nucleic acids tend to partition into the organic phase. If necessary, the chloroform extraction is performed two or three times in order to completely remove impurities from the aqueous layer. To perfect the extraction of nucleic acids, it may be back the organic phase with an aqueous solution which is then added to the above extract. Once purified nucleic acid complexes, you can proceed to precipitation, the last stage of the procedure.

Power Soil may not be the best protocol for soil with low pH (see article from Sagova-Mareckova et al 2008).

For soil with high clay content, you can also have look on the article from Hale and Crowley (2015) who developed a protocol for soil with amended-biochar sand or clay.

Finally, have look on the article from Terrat et al (2012) comparing protocols for different soil (including clay) for metagenomics.

And as previously mentioned, the article from Hurt et al 2014 is a good starting point.

The suggestions the others have made are all good and relevant. However, in your response, what do you mean by "molecular identification of the tree stem and leaves"? Did you mean morphological, rather than "molecular"? If you have stems and leaves can you not also get flowers and fruit (or cones and seeds) and identify this tree from those features? There's also a huge literature on the identification of wood, using anatomical features. Why are you even bothering with the expense and uncertainty of using molecular methods if morphological data will answer your question?

Discovering whether DNA barcodes (or any DNA sequences) can assist with identification requires that you have several reference sequences from all candidate species (e.g. all of those known to be in the flora). Three of the plant barcode markers are chloroplast loci, while ITS2 is nuclear. Visit http://www.boldsystems.org/index.php/resources/handbook?chapter=2_databases.html&section=id_engine to find out more about how to take advantage of the DNA barcode sequences that have already been archived.

Doing good science depends on making use of the tools most appropriate to the problem. Learning to use molecular methods is unlikely to be very productive if you don't take advantage of the knowledge already available from other sources. To this end, you should also have a look at this critical evaluation of how sequence data used in identification should be used: Ross HA, Murugan S, Li WLS. 2008. Testing the reliability of genetic methods of species identification via simulation. Systematic Biology, 57: 216-230.

Thank you for your contribution.....

For ALL molecular reactions you should ONLY use molecular grade reagents. The chemical impurities are not necessarily the issue, but can be. The primary issue is the DNAases, RNAases, proteases and nucleic contaminates. The enzymes will degrade your DNA/RNA and the nucleic acid contaminates may influence your results. The differences between the Laboratory/ACS/ACS plus is based on  purity of chemicals. Molecular grade purity is based on absence of "ases" and DNA/RNA contmainates.

Incubation at 65 degree centigrade in CTAB ensures complete lysis of cells in the suspension. This digest proteins, suspends lipids, and also digest/breakdown celluloid material and make DNA free.  For idea you can refer attached paper.

Colorful DNA pellets do not  imply useless DNA. Some natural dyes provide color at very low concentrations in the DNA  (for instance, those existing in Drosophila's eyes). Have you checked the real impact of the color in the reactions you perform on your DNA?

Dear Luke,

It would be quite informative to have your RNAse preparation protocol and your CTAB extraction protocol, if you are interested in finding how what's happened.

RNAse is usually prepared by boiling at 100C for 10 min because is quite thermoestable, whilst the DNAse gets quickly denatured (see Sambrook Maniatis book, e.g.)

Also, how have you quantified your DNA concentration with/without RNAse treatment? I know it's obvious, but you cannot really distinguish DNA from RNA with spectrophotometry. Therefore, when you RNAse-treat samples you degrade the RNA into nucleotides or small oligos, and depending on how you precipitate afterwards you might not recover that degraded RNA, resulting in a lower nucleic acid concentration in comparison with the non-RNAse treated sample.

Cheers,

Ruben

CTAB has two ends, a lipophilic (cetyl chain) and a hydrophilic-hygroscopic (trimethylammonium + Br^-). When it is crystallized from a protic solvent, a hydrophilic part in the crystal is oriented to surface of the crystal. Then comes the interaction of hydrophilic part with air moisture and the crystal is dissolved. In the crystallization carried out from an apolar solvent the hydrophobic cetyl chains are at the crystal surface and moist air dissolution does not occur.

On the other hand, CTAB is very poorly soluble in nonpolar solvents.

This discrepancy can be solved, so that CTAB is dissolved in a minimum amount of a polar solvent and the solution is then poured into a large excess of non-polar solvent with vigorous stirring (or better under ultrasound action). It is obvious that both solvents must be miscible. For example, the couple EtOH-Et₂O, iPrOH-hexane, EtOAc-MeOH, AcOH-toluene, ...

For DNA extraction I used Qiagen DNeasy blood and tissue mini Kit. The purified DNA was stable at 4°C for some time. So there should be not at all a problem with it. I used bisulfit sequencing and MS-PCR approaches and both worked quite good. You can also use high resolution metling to analyse your samples for methylation, however this shows no status for certain residues

regards

Sven

Thanks everyone.  According to one of my colleagues, the red pigment produced exorbitantly by the fungus Fusarium graminearum on nitrogen poor PDA is the reason why my extraction didn't work.  It get CTAB to work I need to grow it on complete media

I tried clean ups with additional chloroform and phenol cleanup, but it was no use.  Thanks for the article, everyone. Sinai I can't wait to read that simple method.

Cheers,

Tom 

Hii...

Rupak Kumar Sarma...

Please find the paper attached that may fruitful for you...

Best wishes..