Science method

Plant DNA Extraction - Science method

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Greetings to all!!
DNA is isolated from infected cotton leaf.
The image is attached. It looks kind of shearing.
What are possibilities to use it for PCR?
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Hi Dr. Bawankar,
The DNA seems to be smearing significantly, but some remain visible and concentrated near the wells. However, be cautious as overloading the wells with large amounts of DNA can potentially result in smearing. It would help if you considered increasing the agarose percentage. Other factors contributing to smearing, are excessive gel running buffer (more than 5 mm above the gel surface, elevated voltage, and using old buffers, all of which can affect DNA movement on the gel.
Additional RNAse treatment may not be necessary as RNA is unlikely to interfere significantly due to the PCR's staging and cycling conditions. During the denaturation phase of PCR, any remaining RNA will be eliminated from the reaction.
Kind Regards,
Dharmesh
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The QIAmp Fast DNA Stool Mini Kit has two main protocols listed in the handbook, accompanied by some simple notes:
1) Isolation of DNA from Stool for Human DNA Analysis
Lysis conditions in this protocol are optimized to increase the ratio of human DNA to nonhuman DNA.
2) Isolation of DNA from Stool for Pathogen Detection
Lysis conditions in this protocol are optimized to increase the ratio of nonhuman DNA to human DNA.
I'm interested in extracting and amplifying plant DNA from mammalian faecal samples to explore dietary habits. From my reading, it seems that both of these protocols have been utilized interchangeably in various papers to investigate diet, however, I'd appreciate any thoughts on whether one of these protocols might be better than the other for increasing plant DNA yield?
Thanks!
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Well, I think the second method is more appropriate.You can prepare a list of plants fed according to the desired organism and then proceed to extract their DNA and then check the feces and match the extracted DNA. Then you will find out the amount of DNA of the desired plant by using quantification extraction methods. Of course, it is a time-consuming process.
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Genomic DNA, Chloroplast DNA, PCR
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No, u need fresh leaves so that the chances of chloroplast damage are reduced as compared to dry. fresh leaves have more chances of extraction as compared to dry.
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Is it feasible to swap 2-mercaptoethanol with any alternative chemical during plant DNA extraction from sediments?
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I read somewhere that DDT is a suitable alternative. It can be used for most samples except lysis of blood, and epithelial cells.
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One of my colleague is trying to isolate DNA in order to check a bacterial disease in olive. However she couldnt manage to isolate DNA up to now. She tried plant DNA isolation kit and CTAB buffer but after isolation she couldn't see any band in agarose gel. Also spectro results are not good. very limited amount of DNA and very bad purity. Do you have any experience DNA isolation from olive leaves or vein of the olive leaves?
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Sorry for not adding the solution.
We used mericon Sample Preparation and Bacteria Kits(QIAGEN) instead of plant DNA isolation kit and solved the problem.
Also we used only the olive leaf vein not all leaf, because we were looking for a bacterial disease that damages the leaf vein
I hope this solution can help others.
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Hi, I'm trying to isolate total DNA from dried oak leafs using CTAB protocol.
Normally, after the incubation in the lysis buffer (2% CTAB, 100mM Tris, 20mM EDTA, 2.8M NaCl, 1% PVP, 1% BME) and two washes with CI 24:1, I put 500µl of the aqueous fase in a clean microcentrifuge tube, add 500µl of chilled isopropanol + 50 µl of Sodium Acetate and a DNA "cloud" form immediately. Lately the isopropanol mixes poorly with the aqueous fase (some times not mixing at all) and the precipitate doesn't form. Does anyone know what can be happening?
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After adding isopropanol store this sample at -20 for overnight. This will also enhance your DNA concentration.
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I am looking for a good plant DNA Extraction Workstation and a kit for a 96 well plate. I've already heard about the Beckman Biomek Liquid Handling Station, the BioSprint 96 robot, and also the KingFisher™ System. Do you have any experiences with them? Can I use the same workstation to do PCR prep? About the Kits, do you have any experience with Sbeadex® plant kit, Kleargene Plant 96 DNA kit, MagMAX™, or BioSprint 96 DNA Plant Kits? How is the average DNA yield and quality? Do you have any other equipment and kit suggestions for wheat leaf or seed DNA extraction?
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Thank you all for your reply. Actually, I'm looking for equipment and also a kit to do the DNA extraction entirely automated. Do you have any expirience with a equipment or workstation for DNA extraction using magnetic bead-based DNA extraction?
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I have a question about the role of NaCl in the DNA extraction process. So for NaCl concentrations under 0.5M, CTAB and DNA molecules can create complexes. In those concentrations, proteins and other hydrocarbons are still soluble in water except the DNA-CTAB complex. If we raise the concentration of NaCl then the complex of CTAB-DNA will be soluble to water too. Correct me if I'm wrong until now.
So I don't understand something. Why do we use NaCl?
Let's assume that we don't add NaCl. Will the CTAB create complex with DNA or not? As I read CTAB creates ionic bonds with DNA (phosphate groups). So in this situation (without NaCl) CTAB must be able to create complex with DNA too.
So why did we use NaCl? Just to keep proteins and other hydrocarbons soluble in the water or there is something else that NaCl helps in this complex creation?
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CTAB:
· To eliminate contaminants in field samples.
· To disrupt membranes.
· Along with some other chemicals (PVP) CTAB minimize the effect of secondary metabolites.
At a low ionic strength, it precipitates nucleic acid and acidic polysaccharides while under a high ionic strength, it gets bound to the polysaccharides and forms complexes and also inhibits the activation of protein and enzymes (Heikrujam et al., 2020).
NaCl:
· Helps to remove proteins that bind to the DNA and keep the proteins dissolved in the aqueous layer, so they do not precipitate in the alcohol along with the DNA by neutralizing the negative charges (Heikrujam et al., 2020).
Remove the polysaccharides and increase the yield of DNA and prevent the interaction between DNA and polysaccharide (Hamblin., 2009).
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Good day.
Does anyone have any protocol in using disruptor genie for plant tissue lysis? What bead size and amount did you use? How many minutes and rpm?
Thank you very much.
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Check out this PDF. Inside it mentions "Genie disruptor" and plant tissue used as material. Renerio Jr. Pelegrino Gentallan https://fnkprddata.blob.core.windows.net/domestic/data/datasheet/ZYR/D4306.pdf
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Looking expert opinion...
I have collected marine sponge samples and were shadow dried for two weeks. Now the sponge samples are well dried and can be directly powdered by grinding. I would like to study the sponge associated actinobacterial populations (uncultured) from the dried sample rather than fresh sample.
Here comes my doubt,
If we grind and use the sponge powder for metagenomic DNA extraction, does the DNA be damaged/sheared ?
or
can directly use the dried sponge material (without grinding) for metagenomic DNA extraction?
Kindly, some one clarify my doubts.
Thanks in Advance,
Siva
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Hi Sivasankar Palaniappan ! How did the shadow drying method affect the DNA quality of your sponge sample?
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Hi there,
I'm looking for a High Molecular Plant DNA Extraction Kit for subsequent Nanopore sequencing. Does anyone have experience using commercially available kits? Any suggestion/recommendation?
Thank you very much beforehand :)
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I work on extracting DNA from human tissues or blood
Thanks for you
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I've found that my concentration of DNA is less than the expected value.
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These kits yield a maximum of 30ng of gDNA. So it is normal to have a single digit amount. What is important is to check the concentration and 260/280 (1.8~2.0)
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Hey everyone. I collected some leaf tissue samples from the plant Phragmites australis from which I am hoping to extra DNA for sequencing. I will be extracting DNA using Qiagen DNeasy Plant Mini Kits. I was in a rush when storing them, however, and I just placed them in ziploc bags in the freezer at -20oC. They had been kept in the same bags in a cooler while transporting them from the field to the lab.
Is this going to be okay? They've been in there for a few weeks at this point and it may be another few weeks or even a month or two before we will be able to begin lab work. Would it be possible to move them to -70oC now or is it too late? Can they be thawed and dried at room temp in silica gel? Just wondering what my options are here and what I need to take into consideration. At this point, it is too late in the season to collect new tissue samples so this is all that I have to work with.
Thanks in advance!
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In my opinion, you can store the samples at -20 degrees without any doubt...
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Ozodbek Abduraimov
, Thanks dear Ozodbek,
The blow research article has been conducted from this work, and another paper under editing.
"A novel horizontal subsurface flow constructed wetland planted with Typha angustifolia for treatment of polluted water".
Thanks again
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I am preparing CTAB buffer and I read that addition of PVP (Polyvinylpyrrolidone) helps to remove phenolic compounds. Most of the times in protocols there is no additional information about average molecular weight of used PVP, but I found in some, where it was specified that it was PVP 40 (mol wt 40 000) for DNA extraction. But I wanted to know if I can use the same for RNA extraction or should I use PVP 360 (mol wt 360 000)?
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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.
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Dear fellow scientist,
For RNA isolation I use Trizol, however is pH of Trizol between 4- 6 which allow 
DNA to be retained in the organic phase and interface, leaving the RNA in the
aqueous phase?
Greets,
Tse
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Yes, low pH phenol (presence of H+ ions) neutralises the neg charges of the double stranded DNA phosphate backbone. But, because single stranded RNA has unpaired nucleotides exposing negative and positive charges as well as the neg charged backbone, H+ only neutralises the negative charges leaving the RNA polar due to uneven distribution of pos charges across the single stranded molecule.
Once in this acidic environment, the DNA can be precipated in a non-polar solvent such as phenol (or alcohol) where the polar RNA molecules will remain dissolved in the aqueous (polar) environment.
Note, this takes a shot of chloroform to make it happen
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Please provide me practically viable solution.
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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
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I am doing DNA extration from whole saliva using Vivantis DNA extraction kit for Viral nucleic acid purification. After DNA extratcion on PICO drop the values of DNA is 333 microgram per microliter or 423 microgram per microliter, whereas the ratio of A260/A280 is 3.3 or above 3 and ratio of A260/A230 is above 3. Is these values are normal? or high ratios mean?
Is this high ratio of A260/A280 and A260/230 is okay for future PCR analysis?
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Hi there
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I want to isolate the DNA from the chloroplast of Brassica napus. In downstream, I will use that DNA to amplify my target region for cloning purposes. Presently I have isolated whole genomic DNA from it. It was thought that this DNA will contain the chloroplast and mitochondrial DNA in it as well and will be amplified by the chloroplast specific primers. But I am not getting any amplification. Now I want to isolate the chloroplast DNA and give it a try with the PCR. Please suggest for me. Thanks
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Hello!! I'd like to ask one thing. I have isolated Chloroplast from leaves with percoll gradient. Chloroplast, observed under a microscope were very beautiful. After I extracted DNA with a DNA extraction kit and amplified with chloroplast genes. I think that in my chloroplast DNA there was nucleus contamination... how can I obtain a Ch DNA without contaminations???
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I am looking for reports about Roots expanders used in ORC-systems.
Especially when they are used for steam quality <1.0.
Reports about Roots expanders used in water/steam systems are of course also of interest.
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Actually I was looking for expanders having built-in volume ratio = 1.
Having built-in volume ratio = 1 means that the generated power can be high.
The disadvantage is that the adiabatic efficiency is low.
If you will have an expander with optimized efficiency you must decrease the generated power.
Then it's a matter of cost. One might perhaps have to compromise.
If you for example design a machine with say 1 % efficiency below the optimum
you can get out more power from the expander.
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I have extracted DNA from cacao leaf tissue (freeze dried).
I used CTAB buffer (5%) along with 1% B-mercaptoethanol and 3% PVP. I did the DNA extraction overseas and transported it as a pellet to Australia. When they arrived in Australia I stored them at 4 C after resuspending them in TE buffer. However, when I checked the quality on agarose gel surprisingly all of my samples have degraded DNA.
Could anyone pls advice me what can I do to prevent the degradation of the DNA? The downstream application of the DNA will be DarT analysis.
I have attached the gel picture as well.
Regards
Gurpreet
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you can have a look on this article, you will find useful tips to avoid DNA degradation:
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If I have a plant leaf with Alternaria sp. infection. Is there a commercial kit to extract the fungal DNA only? OR the plant DNA ?
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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.
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I am isolated DNA from some folliose lichens, then purified in ethanol at 13000 rpm, after 15 minute drying, pellet doesn't dissolved in elution buffer. I am also using warm water and ( 65 C) elution buffer but pellet doesn't dissolved after 15-20 days. How can i troubleshoot this problem.
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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
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I have to extract DNA from a large number of garlic leaf samples for SSR analysis. I am doing a bead milling technique using SPEX SamplePrep GenoGrinder. My current protocol involves using lyophilized garlic leaf tissue (around 50 mg), two 3.2 mm metal beads, 2 ml tubes, 1200 rpm for 45 sec with pause every 5 sec to rest tubes in ice. My extraction buffer is composed of 3% CTAB, 100 mM Tris-HCl ph 8.0, 20 mM EDTA, 4% PVP, 3% B-me, and 2 M NaCl. My problem is that all of the DNA that I get from my method is sheared/degraded. Is there anyone who has an experience using the bead milling technique for DNA extraction of fragile leaf tissue?
Thank you in advance.
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Dear Grace
Please see the attached file.
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We are working on the functional recovery aspects in response to various phytochemicals. Our targets will be the expression levels of AChR. We want to extract the RNA in muscles but we don't have tissuelyser in our lab. Would anybody advise us the better way for RNA extraction without using tissuelyser please? 
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Hi Yunita, you should add enough liquid N2 to cover the sample and keep frozen during the procedure, you must play attention when you've already little pulverized the sample avoiding too much liquid N2 for the sample spilling. You must keep the resultung pouder frozen until you want add trizol or other buffer for RNBA extraction. Good luck
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It says it is to prevent shearing but does not explain why. Anyone?
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As per your question it seems you are using broad leaf dicot plant leaf for the DNA isolation. For the DNA extraction it is preferred to use the fresh and new tissue for easier and quality DNA extract. The main purpose of avoiding large veins is the complete grinding and homogenization of the tissue. Large veins could hamper the homogenization. Additionally, a minor effect of the veins could also be the presence of comparatively higher plant metabolites/secondary metabolite or other specialized chemical could possibly effect the quality of DNA (rarely seen in daily lab practice).
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As I am trying to extract the DNA from herbarium Fungi and some of them are really old, need some advice to get the best result?
Thanks in advance.
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Let the lysed material overnigth around 40-45 C. But anyway, several times the results are incomplete sequences with several gaps or incongruences, which are hard to align.
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I'm planning to do extractions for metagenomics & metatranscriptomics of insect gut contents. Any other recommendation for quick isolation and separation of DNA and RNA from the same sample would help me. Thanks
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Though I haven't used the kit you mentioned, all DNA/RNA extraction principles are similar. You can have a try.
You should pay attention to preventing your extractive from contaminated and degraded.
Good luck
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Can anyone kindly tell me what amount of rice seedling leaves and root is required for isolation of good quantity of RNA ? Should I weigh the freshly chopped samples before homogenizing with lN2 or after?
Also can anyone please suggest methods for storage of rice seedling root and shoot samples for later RNA isolation? 
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~100mg
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I want to make sure that my plant extract has no fungal contamination
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Really i agreed with dear Oadi Manty ,First you can filter extract with either 0.45 or 0.22 micron millipore , then if want confirmation, you can check with PDA or any medium for Fungi
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The same way as universal primers targeting the 16S rRNA gene are used to detect bacterial DNA, which is the best way to assess the occurrence of plant DNA in soil samples? No need to evaluate diversity or taxonomy, just the occurrence.
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You can look into the maturase K (matK) gene for assessing the occurrence of plants in your sample since it is a plastid gene. I first thought of rbcL but then it can also detect cyanobacteria.
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Hi,
I would like to extract RNA from biofluids, and we have a home-made protocol that I got from a collaborator, that uses Zymo spin columns. However, we have bunches of miRNeasy micro minelute spin columns (Qiagen). Do you know whether those columns are similar in terms of composition, chemistry etc.?
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Hi Iddo Magen,
I am using miRNeasy spin columns for isolating RNA from Saliva and blood plasma/serum. I found quantity and quality quite good. I am using these samples for gene expression studies and I haven't got any problem. About Zymo research I don't have much experience, so I can't give proper opinion although. 
All the best. 
Parwez
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i am working on Gene expression but cant get it done because i am not getting any RNA extracted from my plants. my colleagues says its just because of rapid degradation. what are the main reasons for RNA degradation ?
i am using rice crop..
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Clean the area(where you are working), pipettes with 70% Alcohol and RNAse ZAP solution(I used Invitrogen). The eppendorf,tips,mortar pestle evrything you are using should be DEPC Treated then autoclave it( twice). During the RNA elution steps, carry out the process in ice so that the RNA donot get degraded. After you elute the RNA, perform the experiments in ice only. Keep in -80'c for storage and avoid repeated freeze thaw.
Check your RNA Quality in Gel and also in Nanodrop then proceed ....
All the best.
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DNA will be used for SSR detection in coffee.
Thanks.
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Thank you all!
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I am doing some RNA extraction from tomato leave samples for my gene expression work and by checking the concentration of my RNA extracted on a Nanodrop spectrophotometer, the concentration I got were as high as 1000ng/uL.
Can someone please help?
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I think 1000ng/uL is too high. Samples definitely have DNA contamination. I will suggest you to check DNA contamination by normal PCR using some general primers. so that you can come to know if u have any DNA contamination. If you have DNA contamination do DNAase treatment again. Can you please share 260/280 ratio? if you checked concentration by Nanodrop.
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My student has bundles of bee-collected pollen from a phenology study, and would like to get DNA barcode data from them. We have tried following protocols involving liquid N2, and grinding 0.1g of pollen into "fine powder" with mortar and pestle. It never seems to "powderize" but rather turns into a mushy mess. Yields are very low, between 8 and 12ng/uL. Would appreciate any tips on pollen extraction. Post-(semi-successful) powderization, we use the Qiagen DNEasy plant extraction kit.
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Pollen has a VERY high lipid and protein content.  Try an old school phenol chloroform extraction after grinding in the extraction buffer.
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Hi everyone,
how do you determine the size of genomic DNA? You cannot use a gel because the DNA is too big, any suggestions?
What would you do to determine the size of big DNA extracted from cells without breaking it?
Thanks
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thank you so much for the information, they are all very useful.
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I want to ask that I have isolated DNA from bacterial samples but the problem is that i'm not getting any amplification using the same isolated DNA for PCR I want to give it a proteinase k Treatment again but I don't know the exact amount of proteinase k to be used for 25-30 microlitre of DNA
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I agree the Second part of  Mangal Singh's comment. It could be other reasons. Had you included a positive control in your PCR experiment? The positive control shoul give you a band in normal condition.
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I try already 2 protocols but there are not good enough, the nanodropo and the electrophoresis gel does not give us good results!  
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Hi, Here goes my protocol that I use for fish egg's RNA extraction. I guess, it will help you to extract RNA from micro-algae as well.
Frozen Tissue (can be stored at –80 degree)
               >       50 Zebrafish embryo+600 µl TRIZOL. 
               >       Homogenize by 1ml Syringe plus 23 Gauge needle and mixed by pipetting.
               >       5 min @RT
               >       Add 80 µl chloroform, shake vigorously by hand for 15 sec
               > Centrifuge @12x1000g for 15 min
            Transfer aqueous phase into a fresh 1.5-ml RNA tube
          >  add 5µg Glycogen
               >       + 200 µl isopropyl alcohol,
               >       incubate @RT for 10 min
               >       centrifuge @12000xg 10 min at 4C,
               >       wash pellets with 1ml of 75% ETOH
               >       Centrifuged, 7500Xg for 5 min at 4C
               >       Air dry RNA pellets for 5-10 min
               > Dissolve in RNAse-free water
               >                   “Total RNA Solution”s
               >       yield estimation with A260 and A280
               >       Electrophoresis Check with TAE-Agarose Gel
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How can extract DNA genomic with high purity in fungal with the CTAB method?
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Thaks a lot.
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In order to get a higher concentration of DNA with Molysis extraction kits (Molzym-Coimplete5 and UMD) we are eluting columns with 50 ul, instead of 100 ul as recommended. Can it affect DNA quality?
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The single biggest issue with protein contamination using this method of purification is ensuring complete cellular lysis.  If this and the subsequent wash steps have been performed adequately eluting your sample in a smaller volume, though inevitably concentrating any contaminating proteins, shouldn't be a problem.  When eluting with a smaller than recommended volume I always 1) let the eluent sit on the column for about 5 min before spinning and 2) re-apply the elutent to the column a second time.
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Genomic DNA  extraction using CTAB I use 800 CTAB buffer and 30 microlitres RNAase.I use Maize plant leaf fresh
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Hi Kiran. For this amount of shearing I wouldn't be too concerned. Cheers, Anthony.
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Dear all,
As a newcomer, this is my first time to ask for help about my research projects. please say all you know about my question. Thanks very much.
Now I am busy with the isolation genomic DNA from the chocolate, but the results is very poor, the DNA elution is lightly yellow and no target band can been seen by the agarose gel. How to deal with the though question? Do the every member of the researchgate have a ideal method to resolve the problem?
best wishes
Yunjing Zhang
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Please see following publication on comparative Evaluation of Six Extraction Methods for DNA Quantification and PCR Detection in Cocoa and Cocoa-Derived Products
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I am doing an rna extraction from tomato leafs, using the Trizol protocol. But when I add chloroform, and centrifuged the samples, the hidrofilic portion gets a pink coloring. And the color keeps going in the steps. Any one know what it is? I am afraid that could be Guanidinium thiocyanate, and could be resulting in a low proportion of 260/230. Any information is valid.
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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
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Please I need the most efficient method of isolation of natural dyes from plants.
Any ideas?
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Thank you Michael 
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I was working with some lignans but accidentally I contaminated these ones with a mixture of alkaloids (the ones I said before). So I was thinking on using recrystallization but I wanna know if anyone of you guys has a better idea or maybe some tips to do the recrystallization.
Thank you! 
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I'd expect that under acidic conditions, the lignans would be in the dichloromethane (or whatever you use as an extraction solvent) and thus not available for hydrolysis. 
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I am just into mycorrhiza research. I understand that preserving root samples in ethanol is easy and safer, especially for large samples. I would like to know whether the ethanol (70%) must be rinsed with water before the clearing and staining steps.
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 Yes, we need to remove ethnol and KOH both before proceeding towards staining steps. ...Here is a procedure  entitled A modified procedure for staining roots to detect VA mycorrhizas by R.E. Koske. Mycological Research, Volume 92, Issue 4, June 1989, Pages 486-488,https://doi.org/10.1016/S0953-7562(89)80195-9
Another PDF enclosed to give you fair idea..
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Other colleagues are extracting good quality DNA of other crops with the same chemical. so all chemicals are working good.
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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?
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I want to know which of these options apply during fieldwork.
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Hi Carlos: We do a lot of DNA extraction from plant tissue, so we dry it by using silica or freeze dryer, and its work very well.
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Can the beads be washed with phenol or chloroform, or any other solvent?
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You can use isopropanol, however you risk higher contamination as it is less volatile.
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I need to remove lactophenol cotton blue from samples for PCR - there is an ethanol wash step in DNA extraction protocol, but perhaps another solvent (phenol/chloroform?) should be used?
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Lactophenol cotton blue from sample you can try ethyl acetate chloroform, DMF, benzene in which it has better solubility.
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100 ul barrier tips is common and needed. However 10 ul barrier tips may not be practically needed. Please give your opinion. 
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Hi there,
These are absolutely not necessary for DNA/RNA work. Accurate pipeting, clean and sterile tips and good laboratory practice are enough.
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DNA extraction from a plant leaves rich in phenolic compounds.
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I have a similar protocol for extraction of DNA from plant leaves.  If this protocol doesn't work, you may add PVP to the initial grinding step.
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I have tried following several protocols to extract DNA for my future SSR using primers for PCR amplification (Lodhi et al. 1994; Doyle & Doyle1987), from Sesame leaves.  Interestingly, some samples showing good and clear band (down) however, other samples (upper) are not showing sharp band on my Agarose gel electrophoresis. Does anybody know what is the problem with my samples or which protocol to follow for good quality DNA isolation? I would very appreciate for any comments and guidance
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If your samples are sticky and resinous, I would suggest pre-extracting them with acetone, hexane or chloroform. This won't affect the DNA, but it should remove most of the resinous material. 
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Any one can suggest  a protocol for silica based plant DNA extraction.
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Hi,
I was wondering, if it is possible to extract total RNA and DNA from insect which have been fixed or stored in 70% ethanol at RT and some specimen at 4 degree. The isolated total RNA would be used in RNA sequencing analysis and Whole genome analysis. 
If anyone knows information about the isolated total RNA and DNA, is it working for sequencing and genome analysis or not and what type of ethanolic effects showing on specimen RNA/DNA. I would really appreciate for the help.
Thanks in advance,
Maheshkumar
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Another question for curiosity - What should be approach from DNA - Whole genome sequencing / Metatranscriptome / Whole transcriptome analysis for getting the gene and protein information as well? 
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Has any one extracted DNA from the walnut leaf samples preserved in 70% ethanol? I am worried about the quality of DNA. Can it be used for further analysis like linkage mapping and studying of genetic diversity ? 
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 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
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RNA isolation from leaves of blueberry
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Thank you very much, Mr Ali, I fully got the answer.  
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I isolate genomic DNA from dried soy bean by genomic dna isolation kit. But is it possible to occur that DNA concentration is seen when tested by nano drop but band is not seen when it is run by gel electrophoresis? what does it mean?
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Extracted DNA from soil by using Nucleospin kit..... followed the procedure and even reduced the samples quantity..but still not pure DNA. 
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Thank u all
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I am trying to find any DNA extraction kit that suitable for field usage. It will be better not to use a centrifuge. Does anyone have any suggestion?
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This looks amazing!! My worry is that most of the animal tissues I will be encountered are CITES species. It will be easier to ship the DNA products than the animal tissues... The later one, we will need to go through a bunch of paperwork, and may (and usually) will take months.
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What is best method for total DNA extraction of endophytes from plant tissue? Can i prefer CTAB method for total DNA extraction or any DNA isolation kit? If kit is best then please suggest me kit name.
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I am trying to isolate genomic DNA from blood with DNAzol. I am getting degraded DNA. Solubilized fraction is red in colour.
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Thanks Andrey
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Does anyone know an alternative method to the disruption of seed that does not use liquid nitrogen?
They are very small seeds, beads and blender are not efficient.
Thanks
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Use Quick-DNA™ Plant/Seed Miniprep Kit, it helps as we have isolated genomic DNA of chickpea seeds.
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DNA extraction using CTAB method or a kit.
The sample is either the mesocarp or peel.
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Thank you all
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I am storing leaves in CTAB buffer before I extract the DA from the leaves. How long can I store (and in what conditions) CTAB buffer before I can use it? I know that when the solution begins to crystallise, it should no longer be used. 
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Hi, It had better to store your samples in -80°c freezer before DNA extraction, Since CTAB just solve completely in pH 8 and the temerature above 60°c, it's recommended to use it freshly and preheating just before use to achieve maximum yield. 
Regards
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All proteins, including membrane proteins, will be denatured, but the amino acid chains will be intact.
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Is there anyone have quick DNA extraction protocol?
I use maize as my plant material, and extract DNA by Epicentre' quick extraction kit. But this item is stopped selling now. I am trying to preparing the quick extraction reagent myself. If you know how to prepare the quick  extract buffer, please share it with me. Thanks a lot!!
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A quick and easy way to extract DNA for genotyping is to use Shorty buffer:
0.2 M TRIS/HCl pH 9.0
0.4 M LiCl
25 mM EDTA pH 8.
1% SDS
Grind your sample thoroughly over liquid nitrogen and add 500μl of the shorty buffer and keep grinding until the sample is fully homogenized.
Centrifuge the sample for 10 minutes at full speed (around 13000RPM) to pellet the cellular debris.
Take 350μl of the supernatant (being careful to avoid the pellet) and add to 350μl of isopropanol. The volumes can be adjusted to suit you but always add the same amount of isopropanol as you add supernatant.
Centrifuge for 10 minutes at full speed and discard the supernatant. 
Wash the pellet in 70% ethanol and then leave tube upside-down on tissue paper to dry - the pellet must be dry or it inhibits the re-suspension of the DNA
Resuspend the DNA in 200μl of TE buffer (10mM Tris/HCl pH7,6; 1mM EDTA) place in the fridge overnight and then the following day flick the tube and then you can use the DNA. 
The technique is good for genotyping applications but is quick and crude, if you need pure DNA you will have to clean the extract up on a column. 
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As i tried to extract DNA from fresh plant tissues using kits, or CTAB method DNA was in bad quality.
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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?
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I had already acetolysed the samples for 2 times, but still no success. I'm dealing with dried pollen and flower samples collected from few herbarium.
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Thank you so much.. I will try to modified my method with both of your suggestions.
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What is the actual difference between extraction procedures of plasmid and Genomic DNA 
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The major difference in the extraction procedure for gDNA and pDNA is how we break open the cells to access the cargo and the inherent nature of the length as well as the integrity of both the genetic material. For gDNA, we blindly lyse the cells harshly (chemical or mechanical) to release all the contents and extract the gDNA by PCI separation. But, in the case of extracting Plasmids Alkaline Lysis method is followed wherein mild treatment to cell membrane breaks open the cells. Both the gDNA and the pDNA is denatured into a single strand by the SDS and NaOH in the lysis buffer. The reaction is further neutralized by the addition of any salts of acetate, wherein the plasmid DNA being smaller in size renatures easily, whereas, the gDNA being quite large and complicated remains denatured. In this regard, care should be taken, not to vortex harshly since the genomic DNA can further break and it's easy for small gDNA strands to renature and mix with the plasmid DNA containing aqueous phase.
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I would like to know the major role of ethanol while extracting dna or rna. In all the solvents used, ethanol has to be added first. Why? Please, I need your help
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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.
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Greeting to all researchers!
Can anyone suggest me with a successful procedure for extraction of genomic DNA from herbarium material of Red algae (Rhodophyceae).
So far we are successful in extracting genomic DNA from fresh material by SDS method. Since this group consist of high amount of polysaccharides, we find it difficult to extract DNA.
Thank you all
Swetha
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Hello,
I just want to find out if you have attempted to recover protein concentrate from the moringa leaf using a green extraction system.
Thanks 
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Hi,
I am sorry but proteins were not the target bioactive compounds of our research project. However, some of the techniques that we employed allowed extracting minor amino acids and peptides which are characterized in the manuscripts.
Best,
Celia
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I have Whatman UNIFILTER microplates used for DNA extraction from Arabidopsis thaliana and I would like to know if there is a way to clean and re-use them (they are very expensive).
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I have not tried this microplate filter in particular but I use the following to recycle silica gel for DNA purification (give it a try it should work).
So if it is glass microfibre made from pure borosilicate glass you can recycle it with washing it with plenty of mQH2O, then incubating it overnight in 1M HCl, then washing again with plenty of mQH2O and then drying it out. Then you need to equilibrate the filter with QBT buffer (750mM NaCl • 50 mM MOPS, pH 7.0, 15% isopropanol (v/v), 0.15 % Triton® X-100 (v/v)). You can recycle it many times.
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I already using Powersoil DNA extraction kit but the result from the extraction was not good
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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.
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I've been sing CTAB to try to extract genomic DNA of fungi for whole genome sequencing. I've attached a photo of how most of my samples look. Can you tell me what's going on? I know I have a lot of RNA. I am using purelink RNAse A but I guess it's not working. My question is: 
Do I have genomic DNA? Is it all degraded? The first band of the ladder is 23,130 bp. I'm using DNA-HindIII
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1. Your sample contain huge amount of DNA, if you can dilute this to 1:10 and 1:20 and recheck would be better.
2. DNA is sheared probably due to shaking, pipetting and chemical treatments.
3. You should try RNase treatment on final dissolved DNA. Add 5 uL 20mg/mL RNase in 50 uL DNA prep + 50 uL Nuclease free water. Incubate at room temp for 30 minutes, then reprecipitate by 3M Sodium acetate (5uL) + 1 mL Absolute ethanol.
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