Science method

DNA Extraction - Science method

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Hi everybody,
I'm about to extract HBV-DNA from plasma. I have modified some homemade methods that are regularly used for Human genome extraction. But I could not get enough DNA for positive plasma samples. I also had a problem with a high concentration of proteins in plasma. I tried a high concentration of NaCl (6M), but it couldn't precipitate even 50% of proteins. I just have seen this problem with serum samples. Maybe it is because of the high protein concentration of plasma. By the way, I also don't want to use the phenol/chloroform method.
Who can help me?
Thank you for your consideration in advance
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Phenol/chloroform methodt worked well for me.
This methodology is already well standardized ( )
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Hi everybody
I'm working on an expression gene project for Human Ovum. I have six ova for each sample and have to extract RNA for cDNA synthesis. Can I go ahead with the conventional phenol/chloroform method for RNA extraction? Do I get enough RNA to assess gene expression ?
Thank you for considering my question
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Thank you very much Laura Leighton an Deepdarshan Urs
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As the capsid of viruses is mainly made of proteins, unlike animal cell membranes, can I still use the SDS lysis ( SDS, NaCl, Tris, KCl, MgCl2, EDTA) buffer for viral DNA extraction?
Do you know any protocol for none-column based Viral DNA extraction?
Is it ok to use phenol/chloroform precipitation?
I really appreciate any help you can provide.
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Have you referred to the protocol in the below attached paper?
If not, then you could try this protocol.
Best.
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Hi everybody,
I'm working on a set-up for COVID-19 RNA extraction, and Sodium citrate is needed in the protocol for lysis buffer and precipitation buffer. Do you know any alternative for sodium citrate that can be applied in COVID-19 RNA extraction?
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Thank you Dear Al-kuraishy
how about Sodium Acetate?
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DO HELP ME WITH THE AMPLIFICATION OF TARGET REGION AFTER GENOMIC DNA EXTRACTION
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Seriously, you need to talk with your instructor/PI/mentor. These are basic techniques. Strangers on the internet are not here to design your project (answer your homework?) for you.
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Hello,
Can anyone suggest a good protocol for isolation of somatic cells. The present protocols we are using now are not very satisfactory. Is anyone having good experience in extraction of cells?
Thank You.
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You'll have to specify the organism and tissue type to get any sort of helpful response. Check the methods sections of recent paper and look for commercially available kits.
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We don't have access to glass beads, enzymes, ethanol dry-ice bath (-80 C). We have tried the following protocol for chemical cell lysis, but it was not successful.
  • Pellet 1ml of 24 h. yeast culture and resuspend in 500 ul of Lysis buffer (500 mM NaCl, 400 mM TrisHCl pH 7.5, 50 mM EDTA pH8, 1% SDS). Heat Lysis buffer to 60 C prior to addition.
  • 10 min incubation at room temperature.
  • Add 150 uL of Lysis Buffer ( 60 % potassium acetate, 11.5 % acetic acid and 28.5 ultrapure H20).
  • Centrifuge at 13,500 rpm for 5 min at 25 C.
  • Take supernatant and add equal volume of PCI [ phenol : chloroform : isoamyl alcohol = 25 : 24 : 1].
  • Centrifuge at 4 C for 10 min at 13,500 rpm.
  • Supernatant mixed with equal volume of 100% ethanol. Incubate at -20 C for 2 h.
  • Centrifuge at 4 C for 20 min at 13,500 rpm
  • Wash with 70% ethanol, centrifuge at 13,500 rpm for 20 min at 4 C.
  • Dry the DNA pellet at 42 C
  • Add 20 uL of Elution Buffer
Any idea what went wrong ?
can someone suggest new method or can tell what need to be changed?
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Thank you Hanna Alalam .
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I'm looking for a method to extract DNA from desiccated crocodile scutes to be used in a genomic DNA study. Is there a best method to extract DNA from these tissues?
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This is very good and relevant question. I think same with extraction procedure tissues of animals.
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(tense-urea) protocol explanation please? Any help
Step by step how to perform tense-urea DNA extraction protocol
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Dear Simone,
I enclose a protocol of salt method, which works very well in our lab. It can be used for isolation of DNA from fin of carp and also from other organs (with some adjustments - details inside protocol).
Good luck!
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We collect oral samples of HPV DNA from outpatients clinics, store them in refrigerators overnight and take them to the lab a day later. Is this method of collecting samples ok if we are using the Qiagen DNA micro kit to isolate the DNA from the samples after? How long do the samples stay “fresh” at room temperature in physicians offices to be used with this kit?
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I dont think there is a gold standard here, we typically consider 24 hours as the limit, but try to collect them asap
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Hi,
I wonder if anyone knows how to use sample release reagent from Sansure Biotech ?
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Hi,
The supplier claims that their "sample release reagent" (nucleic acid release technology), can lyse pathogens at room temperature very fast, with no need to heating, centrifuging or replacing tubes, the sample DNA/RNA can be extracted quickly through simple operations. The reagent is applied for the pretreatment of nucleic acid molecules, to release them from specimens, then the released nucleic acids can be used for clinical in vitro diagnosis or for the detection through appropriate apparatus.
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I am extracting DNA of fish samples for DNA Barcoding. The protocol involves an incubation step at 56C. How to do that? Can a Thermostatic Drying Oven be used?
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Would you like to answer my question exactly? I think we both have the same things.
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Does anyone have a protocol or experience with extracting genomic DNA from very small sample volumes of 10-30 microliters?
The sample is a microbial enrichment from a sewage treatment plant.
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Dear Edina,
I have routinely extracted plant DNA from 20 micro litre sample with extraction kit. It could provide good quality DNA and provides effective RAPD and SSR profiling
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I was extracting DNA using Ctab but I am getting Rna n protein contamination along with good DNA please share ur used protocols n at which stage n quantity u r using of proteinase k n rnase A solutions pictures are attached for reference of genomic DNA on 1% agarose gel
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Hi...
I have used RNAseA and Proteinase K enzymatic solutions at the very start of the procedure, along with lysis buffer addition, around 3-5 micro litres and got good quality DNA without any contamination..
You can also try homogenising the tissues in liquid nitrogen instead of manual chopping. This will give you great results!!!
Cheers!!!
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Would beetles trapped in scheerpelz solution (4 Ethanol:2water:2acidic acid:1glycerol) be still suitable for DNA extraction? I cannot find any literature about this.
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I generally do not trap in this solution, but at least in my experience, DNA tends to be tougher than we get it credit for. When you consider the amount of DNA present in an entire specimen, I would think that the yield may be sufficient. That being said, as Owen mentioned, I think your solution is probably harming your ability to extract. When it comes to DNA extraction, I tend to just give it a try to see what I get. If you're detecting something even a single intact copy is likely enough to yield PCR product. Id give it a shot and look at it on a nanodrop. I also like amplifying my DNA yields with universal primers to make sure that the DNA is amplifiable, so you could give that a shot as well.
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I am going to use the Qiagen power soil pro kit to extract DNA from fish faeces. This kit needs a vortex adapter or Qiagen TissuLyser II for the process. My question is, will Omni 4 bead ruptor will meet the requirements. Thank you in advance.
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Very dear friend:
Your server is a cynical investigator, but I am under his command.
Dr. Alvaro Zamudio Tiburcio
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I recently removed some kidney tissues from frozen mountain whitefish for DNA extraction and the tissue was very fragile, almost liquid. Is this common in kidney removals? I am planning to extract DNA using a Qiagen kit, so if anyone has any recommendations for maximum yield, let me know. I am open to new methods.
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Zarna Patel Hello Zarna, thank you for the recommendation! I will carry out lysis with ATL.
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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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After adding resuspension buffer and lysis buffer to the cell pellet, the next step is the addition of neutralisation buffer which requires immediate mixing. The expected outcome is the formation of large, fluffy and white precipitate. Slight delay in mixing results in the formation of small floating precipitate which remains suspended despite doubling of centrifugation duration, resulting in the decrease of A260:A280 eventually.
What are the remedies to solve this issue and improve the DNA purity? The DNA is plasmid DNA. Thank you.
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Honestly, it might be faster to just start over and follow the instructions. I'm sure you have more overnight culture than you needed.
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Hi everyone, I need your help and opinion about my DNA extraction result. I'm using actinomycete, harvested after in 6-7 days so it is not an old culture. I tried twice and still got smear like this, I extracted using kit. If I have to do treatment, what kind of treatment should I use. Thank you in advance for your help.
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That't not how you check DNA yield or quality. Use another method like a nano drop or set up a PCR reaction.
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I'm currently trying to genotype around 60 brains in my lab and I'm hitting a snag in extracting the DNA. I have had no issues with DNA extraction on our newer cases, however, on our old cases that have been sitting in fixative for up to 15 years I can't even get the sample to lysis. Has anyone had this issue before or have any ideas on how to get the sample to lysis? I've tried extending the lysing time as well as muddling the tissue before adding the lysis buffer (from the qiagen micro kit).
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Please go through one of my research which is attached below. The methodology explained in the paper is appropriate for the kind of situation you are in.
Hope this helps you!
Best!
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I will perform dna extraction from feces and kits are very expensive. I saw that extracting DNA from feces and reading PCR is a bit problematic. Has anyone used any adapted protocol? With lizosima or removal of polysaccharides?
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Dear Marilia,
Did you manage to standardize a protocol for the extraction of DNA from feces with Trizol?
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Can anyone help me... I'm trying to purify DNA from whole blood w/ magnetic beads but yield and ratio 260/280 260/230 are very low.
My lysis/binding buffer is made with 4M thiocyanate guanidine, 50mM Tris-HCl, 2% Triton X-100 and 20 mM EDTA. Is there a difference about using SDS, Triton and Tween?
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you may also want to use Sodium deoxycholate to puncture the nuclear membrane for your lysis buffer. use at 1% w/v
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What are the possible reasons for low yield of DNA/ diluted DNA after its extraction from blood other then wrong elution step??
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Hi..
I need a suggestion to isolate animal DNA from tallow or beef fat..
Proceesed tallow doesnt allow recovery of DNA as it has been boiled over 110 deg celcius. Still i need a method to work with a spike or postive control? 
How to separate cells from fat...i have read some literature regarding centrifuge after treating fat with n-Hexane or PBS or CTAB buffer....can any of it help...?
Thanks in advance
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Abhinav Chaudhary, It's been a few years since this question, but I'd be interested to know if you were finally able to extract DNA from tallow samples. My first attempt was to extract DNA from beef fat was using CTAB protocol, but since it did´t work, i`ve been trying some modifications...Still not working.
I would appreciate it if you could share your experience with me. Thanks in advance.
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I am extractioning DNA from blood but my concentrations is low (about 20 ng/ul)..... can you help me? please...
my peotocol is:
1) add 500 ul lysyse buffer and 200 ul blood to a microtube.
2) 20s vortex and 10 min incubation in room temp and then centrifuge (1min/8000rpm).
3) transfer to new microtube.
4) add 100 ul of NaCl and 20s vortex.
5) add 500 ul isopropanol.
6) mix slowly and incubation 10 min in freezer.
7) mix slowly again and transfer to filtration columns and incibation 2 min in room temp.
8) centrifuge (40s/ 10000 rpm)
9) add 500 ul wash buffer and centrifuge (40s/ 10000 rpm)
10) transfer column to new microtube and add 40ul DNase free water pre_heated (60 c).
11) incubation 1 min and centrifuge (1 min/ 10000 rpm)
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Dear Amin Morshedi .The causes are: poor culturing conditions and plasmid propagation, excessive amounts of starting material resulting in insufficient bacterial cell lysis and column overloading. See the link: https://www.qiagen.com/us/resources/faq?id=82d92bd4-125f-4f54-88d1-d90fbe0fb090&lang=en
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Is QIAamp DNA Microbiome Kit the best or there is more better for dna isolation from cervical swabs for 16srRNA
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Which type of kit did you use in dna extraction from fecal sample QIAamp DNA Microbiome Kit or another kit?Lucas Santos@
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1. add RNase to a sample and incubate.
2. add chloroform then centrifuge to obtain the aqueous layer containing DNA.
Does the chloroform deactivate RNase?
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Yes. Performing the RNase treatment prior to phenol: chloroform extraction has the advantage of removing the RNase which is also a protein. There will be an effective denaturation and subsequent removal of RNase in the PC extraction step. Pawarut Narongpun Hope this helps.
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Both myself and a colegue have recently tried this kit and failed to obtain any DNA. I have gone through tech support from NEB and taken on board their suggestions but still the results are completely negative. The same tissue sources produce good quality DNA with Qiagen genomic tips. I would not be persisting with this kit if it were not for the fact that we are trying to span very long repeat regions using the Oxford Nanopore set-up and this kit is the recommended extraction method for their ultra-long read library prep. Are there better alternative out there (I work with a range of tissues including insect, protozoan, annelid and mammalian sources - rarely do I get the luxuary of blood samples or pure tissue culture cells.
Many tahnks in advance for any and all help
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Nanobind HMW DNA Extraction might be an alternative
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I need to extract DNA from the vaginal flora of cows for metagenomic studies. Does anyone know any extraction technique?
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Genetic and functional analysis of the bovine uterine ...
https://www.sciencedirect.com › science › article › p
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Hi., I have been using phenol chloroform extraction protocol for DNA extraction but the solution is turning milky immediately after absolute ethanol precipitation and a low concentration and shearing is observed after running it in the gel. I am not using any vortex in order to avoid shearing , I have checked and changed the extraction buffer too, and have been carefully handling the procedure.
Protocol:(PDF) A modified efficient protocol for DNA extraction in Silkworm (Bombyx mori L.) (researchgate.net)
I am clueless at this point, kindly help me.
Thank You.
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As it's fat tissues, the ethanol can give cloudy/ milky appearance due to the lipids.
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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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You can use sulfhydryl reductant tris(2-
carboxyethyl)phosphine (TCEP) that is an efficient alternative to commonly used DTT or mercaptoethanol.
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We collect blood in the field from wild birds to determine the sex. We have always used the FTA Classic Card, but we bought the FTA Elute Micro Card by mistake, and we didn't have time to replace them.
Is the lab procedure different for the FTA Elute Micro Card? or can the same methods be used to extract and amplify the DNA?
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Both FTA Elute Micro cards and FTA classic card rapidly inactivate organisms, including bloodborne pathogens, and prevent the growth of bacteria and other microorganisms. Both cards allow to capture nucleic acids in one easy step. The major difference is the sample areas for application.
There are no technical differences between these cards for processing samples. FTA Elute Micro cards will cost cheaper than FTA classic card where FTA classic card can store larger volume of samples than FTA Elute Micro cards. Therefore, you can use both of the cards for avian DNA sexing depending on your resource facilities as well as availability.
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Hi, when I DNA extract from sargassum (brown algae), I did not see any band on agarose gel or band is very weak. please help me.
thanks
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Maurissa Sakti .. Could you share the protocol you used to get the best result or at least the good one among all protocols you tried? Maybe someone here could give a suggestion to solve the problem by modifying the solution or some step to make it a better result.
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I've been having issues with extracting a high enough OD for DNA extraction using isopropanol, even though several articles suggested that it's the best and least expensive procedure.
I've been using
2μl of Protein kinase K (10 pmol/μl) and incubated for 1 hour 30 mins at 56°C.
700μl of ammonium acetate (10 mM),
wash it with 70% ethanol in respective orders of the original protocol.
So far, the OD results are around 1.2 - 1.6 (A260/A280).
a) How can I fix this?
b) Second question: Some suggested to refrigerate the extract samples while incubating with isopropanol (up to 30 mins). They've succeeded in some cases but the percentages weren't high. Should I try refrigerate the sample?
Thank you in advances. Any suggestions would be of great help to me.
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By the PCR method (polymerase chains reaction). We can also proceed by sequencing
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As I am currently doing the DNA extraction from Pseudomonas, I'm not able to get any perfect band result. I don't know why. I've optimised the protocol multiple times but still, the band intensity in the gel is very low or negligible.
Composition of lysis buffer for 100ml:
1. 10mM Tris HCl - 0.157g
2. 50mM KCl - 0.37g
3. 0.1% Tween 20 - 50ul
4. 1N NaOH for maintaining pH (8.3)
A procedure that I perform-
1. 50ul lysis buffer in 1.5ml eppendorf and add a touch of bacterial loop colony into it.
2. 99 C water bath for 10min
3. then cool it
4. Centrifuge it at 5000 rpm for 2 min
5. Keep at 4 C.
Alternative procedure-
1. Take 1ml overnight bacterial broth culture (King's B) in 1.5ml eppendorf
2. Centrifuge it at 5000 rpm for 2 min
3. Decant the supernatant
4. add 50ul lysis buffer
5. 99 C water bath for 10min
6. then cool it
7. Centrifuge it at 5000 rpm for 2 min
8. Keep at 4 C.
Can someone please tell if the compositions and the steps are correct or not and also does the order of this composition during mixing affect the buffer in any way?
For running the gel, should I use 5ul of DNA plus 2ul of loading dye? Is it correct?
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First of all you are never going to get a nice band from genomic DNA because it is going to be a population of various sized fragments, so the best you will normally get is is a compact smear.
Secondly the procedure you are using starts with very few cells, so you won't get all that much DNA. It would be plenty for PCR but not to give you a lot of DNA on a gel.
Third, when you have your 99 C step you are denaturing all the genomic DNA and it is not going to renature very efficiently, so the single strand DNA will also be harder to see on a gel. Also the denatured DNA may well aggregate with the cellular debris and you lose much of it. You are likely to get more DNA from your second procedure just because you are using more cells (about 100 fold more).
It really depends upon what you intend to do with the DNA. If the goal is just PCR then don't worry about it, any method is likely to give you enough DNA for PCR. If you are using it for other purposes, then you probably need to preserve it as double stranded DNA (and not boil it).
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Hi,
I´m in the process of troubleshooting for my cloning experiment.
One problem I stumbled across is that the size of my fragments is different before and after gel extraction. I cut a 2271bp and a 1590bp fragment each from a plasmid (I use BamHI+SalI or HindIII+SalI for this, the sizes are as expected). I run the digestion on a 1% agarose gel. After gel extraction (NEB kit) I run 1µl of the purified fragment again on a 1% gel (I use loading buffer containing Gel red). Now both fragments run slower, at about 3500bp and 2500bp respectively.
What could be the problem, maybe the buffer? Did anyone of you face a similar problem in the past?
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I have not seen this particular effect, but I have seen cases where some of the non-ethidium bromide dyes cause even more extreme distortions in fragment mobility. So I would suspect that the combination of Gel-Red and, as Bashiru Garba mentioned, a possible effect of DNA concentration in the lanes is causing this issue. You can try running a smaller amount of your purified fragments or you can run the gel without any added dye and do a post-staining. There may also be some information about such distortions in the product support for the stain you are using.
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I am working on the isolation and identification of ESBL(CTX-M,SHV,TEM) positive isolates.I have been doing troubleshooting for more than two weeks.But still i didn’t get the perfect band for my pcr products.I have done pcr gradient to determine the annealing temperature and used 2.5 microliter EtBr in 2% gel at 80volts,200AMP, 30minutes for electrophoresis.But still i am getting the same type of band over and over again.I have attached my band picture below.I would be very grateful if anybody could help me to find out my problems and help me in settling up my PCR optimization.
Thanks in advance.
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The gel image is not representative one as you did not mention any DNA ladder or the band size you looked for. Simultaneously there needs to use positive control, negative control, no template control in every batch of PCR as well as in gel electrophoresis to validate the PCR. With this image, it is really difficult to interpret as there is no clue to validate the PCR run. Would you please provide the primers' reference?
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We extract DNA with many kits but the concentration is different in each measure, until zero.
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We use Qiagen extraction nuclei acid kit.@
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Hi!
I am about to use 95% formamide, 10mM EDTA buffer to elute biotinalyted DNA from streptavidin bead. So should I just simply add 1ml of 1M EDTA, 4ml of water and 95 ml of formamide to present 100ml of the elution buffer?
Thanks!
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Hi everyone
I'm processing some leaf samples collected in different rivers for shotgun metagenomic sequencing (Illumina NovaSeq 6000).
The company that will sequence my DNA samples (Novogene in UK) requires a 260/280 ratio =1.8-2.0 (no degradation or RNA contamination).
(Novogene webpage:
But I've sent samples in the past (to the same company but for amplicon sequencing) with 260/280 = 1.7-1.8 and they passed the quality control and went full analysis.
Regarding the 260/230 ratio, they do not refer any requirement but my 260/230 ratios are even lower (the lowest is 0.6).
I'm using the DNeasy PowerSoil kit (Quiagen).
So my question is, how low can these ratios be for shotgun metagenomic sequencing?
What's the limit?
I know that I will probably have to clean some samples but I just want to have an idea to help me select the ones I definitely have to clean.
Thanks a lot!!!
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First you have plants sample is Wash carefully and after that extract prepared and then filter the method for the concentration of, removal and enumeration of bacteria in liquid and air to avoid contamination of microorganisms for example fungi and then used other filtration method Sach as centrifugation.
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How can we extract good quality DNA from MGIT tubes manually for Mycobacterium tuberculosis?
kindly suggest!
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Can l use gel extraction kit to remove RNase A or other protein contamination from my plasmid sample using silica membrane spin columns?
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Paul Weber Absolutely you can use that because the principle is that your DNA will bind to the column and rest of the things including metabolites and proteins will be washed away during the washing step. Hence, even just if you have silica columns will do, that way you don't have to waste money on kits. Wash the column after DNA has bound to it with 70% Ethanol or IPA and elute with warm (42 degree C kind of) TE. It should work perfectly. All the best
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Hi!
It is commonly used of silica membrae membrane in DNA extraction column to bind and release DNA. Yet I wonder can I simply use extraction tube to filter and collect ~20um beads (conjugated with DNA), then take out the whole membrane for the downstream reverse transcription, exonuclease and PCR. Will the membrane block or impede the enzyme to access the DNA on spheres? The membrane will be fully immersed in the reaction mix.
Thanks!
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I'm doing dna extraction for bacterial pellets.
I added 40ul proteinase k and 180ul ATL buffer on the pellets first then put the sample in shaker incubater at 56°c for 45 min.
Then adding 170ul AL buffer (then put in 70°c for 10 min.)
Then Adding alcohol and completing using the spin column ..can anyone tell me what maybe the thing I've done wrong? Thank u
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I usually run the DNA for 20-30 min and sometimes 15 min to just have a look on the integrity of the DNA.
I also made a change for my protocol to try to prevent dna degradation.I added a lyzozyme step first and also extended the incubation time of protinease K to 4 hours as i was given an advice that maybe the problem in not completing lysis step..so the result on the gel was better I think
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I use a silica column based RNA extraction kit which includes a guanidinium thiocyanate lysis buffer. The kit was very good and I always got pure RNA with A260/280 higher that 2 and A260/230 higher than 3!! (first figure)
However, after a while -using the same sample- after RNA extraction randomly the A230 becomes negative! and accordingly A260/230 gets negative, too (second figure) .
The blank is the same Elution buffer. And I'm wondering what could be the reason!??
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Most of the time when that happened to us. It was the reagent we used for blanking. What did you blank with? Guanidine will result in a massive increase A230, not reductions. So unless you blank it with something that has a trace amount of guanidine (or any other chemical that could interfere there) then measure your pure sample, you will have negative 230.
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Hey everybody!
I have difficult times isolation DNA from a Sabourd agar T. rubrum samples.
I take around 100-300 mg of material, scrape it from agar and cut my samples with a scalpel for a small fragments, then I tried incubating them with proteinase K for 2/3 hours, heating samples to 95C for 30 min and beadbeating 3x with 2 mm beads for 5 minutes.
When I tried to isolate the DNA on Zymo kit columns I have at best only 10 to 15 ng/uL DNA disolved in 26 uL. That is too little for anything meaningful. Anybody has any ideas for a working protocol? I still think that my samples are not lysed properly and that is the issue.
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The fungus Trichophyton rubrum is one of the important dermatophyte fungi. It grows well on the Sabourd Dextrose Agar (SDA) with adding cycloheximide to prevent the other fungi for growing.
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Hello everyone
I have leaf samples (+/- 50g) collected from different streams for shotgun metagenomic sequencing and I want to know if there is a way of selectively extract fungal DNA from the leaves without having "host" DNA contamination (leaves DNA).
When leaves fall into the streams they are usually colonized by terrestrial fungi and bacteria but they are promptly replaced by aquatic fungi (constituting more than 90% of the total microbial biomass). These fungi are ubiquitous and have morphological and physiological adaptations that dictate their success as colonizers and decomposers of leaf litter. These features allows them to grow within the leaf structure so washing or sonicating the leaves will not anything.
Taking this into account, my goal is to extract fungal DNA from the leaves but with minor contribution of leaves DNA to assure maximum microbial annotation/detection.
Thanks!!!
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You must wash the plant sample carefully, then prepare the extract and filtrate it by using Millipore filter to avoid the contamination with microorganisms such as fungi, after that you can use filtration-centrifugation method.
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Hi,
I am using the Qubit dsDNA HS Assay Kit with the Qubit 4.
However, I am noticing that I am getting different readings from my standards between batches.
As per the instructions, I make new standards with every batch. I've noticed Standard 1 is relatively consistent at a lower concentration standard ~30 RFU (raw fluorescence units). However, Standard 2, the higher concentration standard, varies significantly and impacts the readings I get between batches.
For instance, in a few batches, Standard 2 was about ~11,000-16,000 RFU, while in other batches, it was 24,000-29000 RFU).
I've had instances where I've double-checked the same sample, and when standard 2 was around 11,000 RFU, the readings were too high- out of range, but when Standard 2 was at a higher level ~24,000-29,000 RFU, the sample concentration was around 44ug/ul.
.
I have contacted the company, who said it should be working as long as Standard 2 is 50 times greater than Standard 1. But I am noticing differences between batches.
I don't know which RFU is correct for standard 2. I was wondering if anyone could share their typical readings for Standard 2? or any potential trouble-shooting suggestions?
I really appreciate any help you can provide.
Cheers,
Jordan
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I have been using Qubit 1x dsDNA HS Assay Kit on Qubit 4 fluorometer for DNA, PCR amplicon, and Nanopore sequencing library quantification with no issue. I always found consistent RFU range for both standards.
What I follow: After adding 10 microleter of each standard and 190 micro working solution, I mix them by vortexing 2-3s and leave them at room temperature for 2 minutes. Then I take readings. I never found discrepancies.
Follow kit's expiry date, storage condition, thaw properly at room temperature, pipette precisely, mix well, incubate 2 minutes, and take readings. Hope for the best..
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CTAB Extraction Buffer (1.4 mmol m-3 NaCl, 20 mol m-3 ethylene diamine tetra-acetic acid (EDTA), 100 mol m-3 Tris-HCl (pH 8.0) and 0.2% (w/v) beta-mercaptoethanol)is not working. Any suggestions?
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They are many good silica-based column kits such as NucleoSpin Plant II (M-NAGEL), DNeasy Plant Mini Kit (QIAGEN)...
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After running the PCR product on gel if the band falls in between two bands of the DNA ladder. How can we get the exact size of the PCR product?
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Hi Iqra, in this case, if you want the exact size you have to sequence your PCR product using the same primers . Otherwise, with a ruler and 2 ladders from each side you can estimate the size of your product.
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Hello,
if have the following issue at hand: Imagine I have attached DNA to magnetic beads (MagPrep) using the PEG/NaCl solution and washed the DNA with ethanol. I will now suspend the beads in a run buffer and magnetise them again. Instead of using an elution buffer I will apply electric current for electrophoresis right away. Will the DNA leave the beads just by electric force?
I read it is possible to open a streptavidin/biotin connection via electric current and would like to copy this method for unbiotinylated DNA (and maybe later for RNA) on carboxylated beads.
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The magnetic field generated by electrophoretic current is negligible. The electric field is too low (~ 1 uV/nm) to induce immediate quantitative release of the DNA from the beads. Most likely , DNA will slowly leach
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I'm looking for some standardized protocol for isolation of dna from cordyceps sinensis for further use in genome sequencing. Suggest if any ??
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The Qiagen DNA extraction kit works great for fungi. Just make sure that you grind it really well to disrupt the cell walls.
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I need a Protocol for DNA Extraction.
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Hi Anosh Saleem, to prepare 100 ml volume of TE buffer:
In a 100 ml bottle, add 1 ml of Tris HCL (1M, pH 8) plus 200 µl of EDTA (0.5 M, pH 8), and complete to 100 ml with water (molecular grade). Store at RT.
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So, I’ve been trying to do midiprep to extract some plasmids for transfection and for transduction, using the PureLink™ HiPure Plasmid Midiprep Kit. However, I’ve been getting inconsistent yields ranging from 200 ng/uL - 800 ng/uL. On good days, I do get yields of about 1000 ng/uL. To be exact, I’m extracting shRNA plasmids from E. coli grown in 100 mL LB broth supplemented with 100 ug/mL ampicillin.
I’m not sure if there’s anything I’m supposed to tweak from the protocol in the kit. In my previous lab, I did a lot of miniprep and always had reasonable yields. Based on my experience, we had to tweak certain steps to improve yields in miniprep. (E.g. Drying column in 55 degrees Celsius)
I started doing midiprep for the first time in April this year and was advised to tweak some of the steps in the kit (e.g. overnight precipitation of eluate in isopropanol). However, my yields were still sporadic and low. I’m just wondering... Is/Are there any step(s) stated in the kit that anyone has tweaked to get better yield?
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1. did you inoculate the colony in a small LB volume (~3ml) overnight, then add the culture to the larger volume (~100ml) the next day for midi?
2. which e.coli that you use?
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I'm considering both the Omega EZNA Blood kit and the Qiagen DNeasy Blood and Tissue kit to extract DNA from avian blood samples. Avian blood is nucleated so I shouldn't have any issues with yield.
My samples are stored in multiple ways which makes things difficult:
1. Whole blood in 100% ethanol (clotted)
2. Whole blood frozen (not yet sure if clotted)
3. Red blood cells on Whatman #903 cards
Regardless of the kit, I'm going to have to got through a preliminary anticoagulation step (if you have recommendations for this, please feel free to share! I have a few methods to try but haven't started yet, so I'm not sure how successful I'll be).
Does anyone have experience with one of these kits or both? I'm extracting DNA for use in a ddRAD study for population genetics so I need high-quality purified DNA. The Omega kit is substantially cheaper than the Qiagen kit. I have used the Omega kit for other extractions (where the quality is less of an issue) with success. I also found one paper that compared these kits and was favorable to the Omega kit (https://journals.asm.org/doi/10.1128/JCM.01894-17?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed&).
Finally, I know there are kits to extract DNA from Whatman FTA cards, are these strictly necessary? I was able to successful extract DNA from a card for genetic sexing using the Omega kit but the quality of the DNA is less important for that use.
I'd appreciate feedback on anyone's experience!
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I worked for a long time with Qiagen DNeasy but also with NucleoSpin Blood (MACHEREY-NAGEL) without any problem. Please take a look at the attached pdf (chap.16), you will find a detailed comparison across different techniques.
For the anticoagulant, heparin should be avoided because it inhibits PCR (cation chelation, Mg ++), EDTA is often preferred.
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I prepared a 24:1 mixture of chloroform and isoamyl alcohol to make 100 ml solution. So I added the 24 ml chloroform and 1ml isoamyl alcohol and together to them added the vol 75ml with dd water. What can be the reson for the two layers formed and troubleshooting for it !!
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Hi,
you should not add any water to the preparation. The two phases are formed by the organic (chloroform) and aqueous (water) liquids. Thus use only 24ml chloroform and 1ml isoamyl alcohol.
Best,
Norman
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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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I am developing a method to detect insect DNA in food samples via DNA metabarcoding.
I already designed primers (1 forward and 1 revers) for an amplicon of ca. 200 bp length that bind to mitochondrial insect DNA. Right now I am testing those primers in PCR to find the right temperature and conditions. I consider amplification curve and melting temperature from PCR and also bands on an agarose gel of all DNA-samples. All insect samples work well, but i have a quite unusual problem with honey bees:
They have a band at 200 bp on agarose gel, and there also is a melting peak at about 78°C. This is as expected and also like all other insect samples. But there is a difference: There are no amplification curves of honey bees in PCR.
I already tried cleaning the DNA extract with magnetic beads, that didn't help.
Additional information: I use EvaGreen as a flourescence dye in PCR.
Does anyone have an idea what could be the issue or what i could try to solve it?
I am happy to give more information about the conditions, if needed.
Thank you in advance.
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I would call tech help at bio-rad. Are the bees all run on their own assay or do you have any results from a plate of some bees and some other amplimer showing this result or are all of the negative results on bee only plates?. If you have a bee pcr band amplified then I agree with Katie A Burnette then this looks like a technical problem possibly something has changed the exes on the graphing so the curves cannot be seen for your bee assay or you have found a way of hiding the display for these samples so the data may be there but just not showing
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I want to avoid ethanol as it is known to hinder downstream amplification process. Also, I am in a process of developing an automated diagnosis of ZIKA and would want to avoid phenol/chloroform and other chaotropic agents which are toxic. This is because my project is aimed at developing a point of care diagnostic device for on field trials in disease endemic areas and would want to use a non-toxic reagent as my wash buffer. Until now, my results by using Nuclisens Wash Buffers as well as 70% and 91% ethanol as wash buffers have been encouraging but I feel that ethanol hinders the downstream amplification step and would want some alternative if possible. Any suggestions?
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Chaitanya Parikh , have you found out any wash buffers that does not need any ethanol in it for washing the magnetic beads? Please let me know if you have solved your problem.
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I boil 1.5g agarose in 100ml TAE 1x in three bursts of 30 seconds, swirling in the middle but a lot of bubbles remain
I have to remove by pushing them to the side when I pour the gel
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Is your agarose actually boiling after heating. If the gel mix is just very hot the bubbles may be air being degassed from the buffer. Actually boiling the gel will degas the mixture and will minimise bubbling
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I need an appropriate protocol to extract mitochondrial DNA from any sample for forensical purposes, which would help provide an accurate analysis. If possible, kindly mention any easy way to develop a mitochondrial DNA isolation kit. Thank you.
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If you want to amplify using mitochondrial primers for analysis you need not specifically isolate mit.DNA, even DNA isolated using normal procedure also useful for amplifying mitochndrial primers. You can check my publication availabe in RG. May I know excatly what are your plans/working!!!
Good Luck!!!
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To all my network.
in our lab. we are looking for one QIAamp PowerFecal DNA Kit, Ref. No. 12830-50. The kit has been discontinued and we need to run few more samples for an important experiment.
Any of you could help us on finding one of this kit?
Thanks in advance for your help
Best,
Giuseppe
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Bassam MS Al-Musawi we have contacted qiagen and they have proposed us the PRO version of the kit. Of course the results with the normal kit and the PRO are not comparable, that's why we would like to run all the samples with the same type of kit for DNA extraction. Anyway thanks for checking in your lab. if you had some of them
Best regards
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I have environmental DNA samples that were filtered through cellulose-nitrate paper and extracted using Phenol-Chloroform.
Samples were collected from small and ephemeral waters, and thus have high environmental contaminants (tannins, humics, etc.).
Tissue-derived DNA extracted with Phenol-Chloroform are not inhibited, so I suspect residual phenol is not a culprit. Besides, samples are washed with ethanol during the extraction after the phenol steps.
qPCR is conducted with SYBR Green.
What I have tried:
Dilution - I ran a dilution series. At 1:100, almost all samples are still inhibited. At 1:500, almost all samples are too low in concentration and fall under detection threshold. 1:200 seems a fair compromise, but some samples are still inhibited and some are too low concentration.
BSA/DMSO - Using both, samples are uninhibited in standard PCR. However, BSA seems to interfere with SYBR dye. DMSO alone does nothing.
Zymo One-Step PCR Inhibitor Removal Kit - this kit appears to have introduced inhibition to samples which were not previously inhibited (see attachments)!
Attachments (all samples are expected or confirmed positives, standards are green/yellow): 
DilAmpPlot (top) - samples diluted 1:200
DCCAmpPlot (middle) - samples processed using Zymo DNA Clean & Concentrator kit (does not remove inhibitors)
OSAmpPlot (bottom) - samples process using Zymo One-Step PCR Inhibitor Removal Kit
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Yes, I imagine that could certainly be a concern. For me, I was more interested in qPCR as being a more sensitive method of detection, but less interested in quantitation. I categorized samples presence-absence, based on critical threshold (which seems intrinsically subjective). With dilution, I had to accept the possibility of a few false negatives, but those would be in my most unreliable samples, anyway. If you are interested in absolute quantities, especially on the lower end of the spectrum, I'm not sure how to reckon with your concern, which I believe to be valid. I never had to consider those issues in my work. I know there were a handful of studies in the time I was doing my research that dealt in quantities, and I imagine there have been many more since. I don't know if any also dealt in PCR inhibition, so I wouldn't know of any good leads to point you towards.
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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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Yes, you can definitely store them. -20 -80 and dry should all work. Dry samples can be a bit tricky to isolate from but are very easy to store. You just want to avoid stuff thawing or getting wet.
On a storage note, plastic bags get very brittle at -80, it is easy to have bags break and spill, you may want to switch to tubes
If you are collecting these samples again I would suggest having duplicates (or more) of each plant and to store them in different locations (like different freezers, or one dried and one frozen). That way you will have a backup!
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I have been using the Phenol-chloroform co-extraction method (Griffiths 2000) to extract the DNA/RNA from anaerobic sludges. After the precipitation with Sodium Acetate and Iso-propanol and washing with 70% Ethanol, then air dry. I found it is very difficult to redissolve the pellet into water. As a consequence, the qubit readings with those samples were very variable. I have tried to dilute the samples and qubit again, the concentration didn't decrease linearly so that the DNA/RNA was not fully dissolved. Anyone knows how to solve this problem without jeopardising the quality of the DNA/RNA.
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Two things may help : 1) avoid drying the pellet too much; air drying is fine, but just leave the pellet to dry until it is slightly moist: most of the ethanol will have evaporated; 2) leave the pellet in water (better Tris 10 mM pH 8 + 1 mM EDTA) for several minutes at 37 or even 60 °C (with the lid closed, to avoid evaporation). Some triturating of the pellet by aspirating up and down with a Pipetman may help, but don't overdo it, lest mechanical shearing should damage the DNA
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I want to extract bacterial DNA from whole blood and there is no need to seprarate bacterial DNA from human DNA, I have not find a appropiate protocol.
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Hi Jianan Wang, could you share with me how you volved that issue. I have the same Problem, se used the QIAamp DNA mini kit, but we did not get good results.
Could you please share with me which kit did you use or which protocol. Thanks
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I am looking for a 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? Do you have any other equipment and kit suggestions for wheat leaf or seed DNA extraction?
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It can depend from how money do you have. There is huge difference in price between fully robotic systems and Kingfisher flex system. We have later one in our lab and it runs well. I can not say about other instruments. It is open system so you can use Plant extraction kits from different producers.
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For DNA extractions we collect young leaves and freeze drie to preserve them for a long time. Due to a storm there was a leakage in the building and the freeze dried leaves are now wet from the rain. Can I freeze drie the leaves again? And are the leaves still usable for DNA extractions?
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DNA is really very stable. I would expect your material to give good amounts of dna after drying again. The second drying is necessary to avoid mould and bacterial degradation of the dna but I think your yields will be fine. There may be contamination issues if the samples got so wet that dampness has spread from one sample into another sample but that is a different issue
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Hello RG,
I have been doing PFGE from yeast, S. cerevisiae, but have always done them in agarose plugs. Does anyone have experience using a liquid DNA extraction for PFGE? Would you be able to guide me to a protocol, I haven't found any during my search.
thanks
-
Matt
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You may use abrasion and maybe sonication, the liquid may be due to the cintents... But not for the technique?
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Hey everyone!
I did a DNA extraction of the hair by the phenol/chloroform technique, after that, I measured them in Nanodrop and obtained good results. To confirm these results I decided to take the Lambda in agarose gel, but when I did that I didn't get any results.
I added a picture of the gel below where you can see: the first marking (that would be the lambda), then comes a large blank part (where the samples should be), and finally a control sample of another animal that was marked.
Does anyone has been through the same problem and know what may have happened?
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Also a possibility that your Nanodrop "good results" can be misleading. Once I faced a similar challenge with a collaborator: they absolutely 100% believed they had put a bunch of DNA extracts on a plate -- sent to us for some DNA assays. Our lab kept getting 'zero' results, like your gel. Turned out the OD measurements were somehow fouled by a contaminant from their extraction process -- never figured out what the chemical was, but it had a UV spectrum enough like DNA to fool the collaborators, and fool us.
I suggest 2 hopefully quick trouble-shooting tests:
1 = Run PCR on a gene that you know for sure will amplify.
2 = I have since occasionally measured a spectrum that absolutely looks like DNA, but the sample is literally just pure water left to soak in a clean new plastic microfuge tube --> some plastics can release a surprising amount of contaminant that absorb in 250-300nm. Very easy to test: let some water (or your extraction buffer) soak for a few days in the exact same tubes you are using for your extractions, then test in Nanodrop. It sounds 'silly' but you might be very surprised. If nothing else, this is a very easy and cheap test.
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I'm in the market to buy a DNA extraction robot, and would really appreciate any suggestions/experience/advice.
With the projects we recently landed we're expecting to process on average about 3000-5000 noninvasive samples per year (scats, urine, saliva - all taken from the environment not from the animal directly). DNA extraction is a total bottleneck in our lab, it's difficult to do quality control when hand-extracting (sample mixup, pippetting errors...) and is too labor intensive (hence expensive) and slow.
I'm not too keen on the magnetic beads technology (tested some machines, didn't like them) and I'd like something that could automate regular spin column (silica membrane) extraction. QiaCube from Qiagen seems an option, but it only does 12 samples at a time. I'm looking at about 100 samples per day throughput, and can spend about 30,000€ on this (well, 40,000€ tops). Contamination prevention is critical with noninvasive sampling applications. 
I'd really appreciate any help with this.
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I am also eager to hear your thoughts on the robot comparison! Can you share? Do you think bead technology would work with eDNA samples?
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Is it possible to extract enough DNA from one unique Apicomplexan oocyst ?
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'Enough DNA' for what? There are kits optimised for DNA isolation from a single cell - what is your downstream application?
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I am working with FFPE human tissues (prostate cancer and normal) for DNA extraction on whole mount slides. I have unstained slides along with its corresponding HE slides. These tissue samples have already been through formalin fixation and paraffin embedding. I did tissue macrodissection and will be extracting DNA from them. Currently I have the FFPE scrapings stored in 70% ethanol in a 4 degree Celsius freezer. It has been stored for a month, due to a kit that was backordered. Are these samples still okay?
thank you!
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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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We use Cl:IAA 24:1 for DNA extraction cleanup step. It would be faster and easier to have this made up ahead of time than to make it each extraction set. It is possible to buy the premixed solution but more economical to make it ourselves. Does anyone have experience with making this solution and keeping it for a week or two? Do you use a stabilizer?
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Dear Heidi McKee many thanks indeed for your very interesting technical question. I see no major problem is you freshly prepare your chloroform and isoamyl alcohol (24:1) mixture yourself and then store it in a brown bottle at room temperature. As you mentioned already in your original question, this mixture is also commercially available. When you buy it form a supplier like Merck or Sigma Aldrich it is stabilized with 0.6-1.0% of ethanol:
Particularly the chloroform in your mixture does not have an endless shelf-life. It must be stabilized with ethanol in oder to avoid the formation of decomposition products such as highly toxic phosgene (O=CCl2) and HCl:
However, the stabilizer is required to store the chloroform for months or even years. When you want to keep your solvent mixture for just a week or two I suggest that there is no need to use a stabilizer. Just use a brown bottle because light can accelerate possible decomposition reactions.
Good luck with your experiments! 😊