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Questions tagged [dna-isolation]

The laboratory technique involving the extraction of DNA from a cell in a pure form.

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Would there be any particular reason to choose strawberries for this DNA extraction tutorial?

The NileRed video What is DNA and what does it taste like? | Edible Chem shows the extraction of a surprisingly large amount of DNA from some strawberries. At 02:22 ...
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Why pellet and resuspend E. coli for plasmid prep

For maxipreps, cant you just add all the stuff that would be in P1 (RNase A, EDTA) then just add P2? Because if we pellet cells then resuspend theres got to be a reason, right? Is it slats and other ...
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UV-lamp, DNA and RNA extraction lab

I'm equipping a lab room (~3x5 meters) that's supposed to be used for DNA and RNA extractions. I know about the basic safety precautions when working with RNA and DNA extraction (fresh gloves, ...
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530 views

Microsatellites and Minisatellites: Which of these form the basis of DNA fingerprinting?

I'm in a fix. Prepare yourself for a long read We've just learned about minisatellites and microsatellites at class (okay, by "learned", I mean we were told their definitions and essentially ...
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217 views

Why does DNA need to be pure for restriction enzymes to act on it?

My textbook states: “In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules. Therefore DNA isolated from other chemicals of the protoplasm like ...
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195 views

How were the first genes isolated from genomic or cDNA libraries without already knowing a hybridization probe?

Let us go back in time to around 1975. It is my understanding that at that point (or at least by the end of the decade), both genomic and cDNA libraries had been created for a few organisms, e.g. ...
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In genomic DNA extraction, is it possible that too much CTAB hinders subsequent DNA precipitation with isopropanol?

I'm trying a genomic DNA extraction protocol that uses 4% CTAB, 3M NaCl, 2.5% PVP-40, 20mM EDTA (pH 8), 100mM Tris-HCl (pH 8) and 3% beta-mercaptoethanol as lysis buffer. I incubate at 65oC for 60 - ...
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Is there a method to isolate half of the chromosomes of a cell?

Each cell has 2N number of chromosomes, I wonder if currently there is a technique allows isolation of N number of chromosomes out ?
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279 views

Why are large amounts of DNA required for analysis?

I know PCR is used to create large amounts of copies of genes and that it's a scientific breakthrough but want to understand why. Why do we need large amounts of DNA to be able to analyse it, to ...
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326 views

Lysis step during DNA purification

I am currently studying DNA extraction from various bio-originated samples. I am new to this field, and have learned how commercial DNA extraction kits work for bacteria. I understand that the very ...
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1answer
55 views

Detecting nuclear DNA in suspension of mitochondria

Is there a way to detect nuclear DNA in a suspension of mitochondria extracted from leukocytes? I need to make sure there is no nuclear DNA in the suspension before extracting mtDNA from the ...
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208 views

During the process of plasmid isolation, how can we be sure that only plasmid DNA has been isolated and there is no chromosomal DNA?

We separate the membrane and all other cell components, but how is it that only plasmid DNA is isolated and not chromosomal DNA?
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DNA extraction from plants and algae don't use phenol. Why?

I don't know if here is the correct place to make this question, but I've been noticing that protocols for the extraction of photosynthetic organisms DNA don't use phenol. Only CTAB / Chloroform. Why?...
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Why is a screening process required in selection from a genomic DNA library?

After the process of construction we go for screening process of selecting a recombinant host. Which includes: DNA hybridisation Colony hybridisation Random primer labeling Nucleotide ...
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569 views

Separation of smaller DNA fragments and larger fragments without using gel electrophoresis?

For an upcoming experiment I would have two kinds of DNA fragments in a sample after a particular reaction step: First there would be fragments of a length of approx. 170 bps and then there would be ...
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How to improve A260/230 ratio?

I am extracting DNA from cicada exuviae using ethanol precipitation combined with either NaCl 6M or Ammonium acetate. After DNA extraction, my samples have quite low A260/230 ratio such as in the ...
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697 views

Genomic DNA extraction protocol by salting out with NaCl, how to improve yield

In these papers A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies A simple salting out procedure for extracting DNA from human nucleated cells Modified salting-...
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5k views

How long can one leave sample in lysis buffer for DNA extraction?

I am trying to do some DNA extraction for my professor, and I make a lysis buffer with proteinase K. I did not have the time to continue the procedure so the hair and the buffer have been sitting ...
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How to cut out a specific named gene from plant DNA

Suppose one has extracted RNA from a plant, converted it to the corresponding cDNA & amplified it but now wants to cut out a particular, already-sequenced gene out from it, how does one proceed? ...
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663 views

Purifying large amounts of PCR product

I have had trouble purifying very large quantities of pure PCR product. We are using these as templates for the reconstitution of nucleosomes and I require hundreds of micrograms to milligrams of ...
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4k views

0.1 M sodium citrate in 10% ethanol and DNA solubility

I understand how DNA precipitation works in the presence of salt (such as 0.3 M sodium acetate or 0.2 M sodium chloride) and alcohol (30 ~ 50 % isopropanol or 60 ~ 75% ethanol) However, in the Trizol ...
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What evidence is there that common PCR DNA purification kits can isolate DNA from pathogens engulfed in macrophages?

I have previously asked a more general question about how PCR sequencing reaches all possible DNA targets here. I want to ask more specifically now: what evidence is there that common PCR DNA ...
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118 views

Function of NaCl in yeast shuttle prep of plasmids

What is the role of NaCl in plasmid isolation from yeast cells using plasmid rescue solution? If the basic theory behind the method is alkaline lysis, then we is NaCl used and not NaOH?
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82 views

Is there any possible way to take a DNA test without using blood in humans?

Is there any possible way to take a DNA test without the need to draw blood in humans? Any information will be useful for me.
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Avoid Blood lyse color remain in DNA extraction

I tryed several times to extract DNA from freezed blood sample whit Qiagene kit but it gave very small amount of product due to good purity then I try extract manually by using CTAB PVP method and ...
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4k views

How long is saliva viable?

Does anyone know if saliva can stay viable for about 5 days, before it gets suspended into a DNA genealogy vial for testing? Background: My brother wants to do a DNA genealogy test at a US testing ...
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596 views

method for DNA extraction for human mucus(nose) or earwax?

What is a simple method for extracting human DNA from nose mucus or earwax? I tried googling for methods for DNA extraction for human nose mucus or earwax and couldn't find any. I assumed that if one ...
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2k views

How much salt [NaCl] is too much in DNA precipitation?

In DNA extractions, how much is too much salt in a CTAB extraction buffer? Protocols hover around 2.5 molar; if you go over this (e.g. 25 molar), will you saturate your solution, and precipitate the ...
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503 views

How bad is ethidium bromide in your plasmid for downstream applications?

I want to transfect Adipose Derived stem cells using a clone of my own. My problem is that I get a lot of contamination (not only genomic, but due to the nature of the technique, I get undesired ...
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853 views

Explanation of protocol in DNA extraction experiment

In the Materials and Methods section of this paper on DNA extraction and analysis from hair I do not understand some parts of their protocol methods. In particular: "The digested hair solution was ...
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1answer
265 views

From a computer science perspective, how is DNA compared for various purposes?

I am very interested in privacy preserving technolgies, such as Microsoft PINQ and would like to see if this is applicable to DNA comparison. Given that I don't have a background in biology, I ...
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361 views

Can these “Promega Wizard genomic DNA Purification kit” be used for samples not stated directly?

I am not sure if this is a duplicate of my previous question, it is somewhat related, so sorry if I misunderstood. The question is, can i use the above kit for samples like hair, fingernails, etc? ...
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693 views

DNA extraction methods for hair?

TLDR: Can anyone state a extraction and isolation method(s) for genomic DNA for hair that will be used for PCR, in detail is preferable since I am a novice. I tried googling for DNA extraction ...
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2k views

Extracting cell-free DNA

Has anybody had any success in extracting cell free DNA from plasma without using expensive kits? I've already spent a lot of my own blood trying to use standard phenol/chloroform methods with very ...
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7k views

How to make µg/ml concentrations of proteinase-K?

How does one prepare concentrations in the mass/volume (weight/volume) form, for substances like nucleic acids or in this case, proteinase? A detailed example would be helpful. I need to prepare ...
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1k views

How does the size of insert affects the rate of Homologous Recombination in yeast?

When performing genetic knockouts in yeast using homologous recombination to replace a target gene sequence via a vector DNA, does the region between the flanking regions in the vector have to be the ...
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1k views

What is the most critical step of plasmid purification?

During the process of plasmid purification and quantification, which step is the most critical to the success of the process?
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2k views

Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?

Which buffer is best for DNA Electrophoresis and which is best for Protein to be have a sharp bond? Considering a higher electrical conductivity compared to TAE & TBE and the generation of less ...
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1answer
79 views

Transcription rate expressed in microarray per hour

This article gives measurement of transcription rate and the unit they're using is microarray per hour. For example, at 27°C the average expression of their genes is 236.1 microarray per hour (page ...
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329 views

DNA extraction from oil-containing seeds

Which protocol of DNA extraction from plant oil-containing seeds take fast and good result in purity that can use in RT-PCR(Real Time polymerase chain reaction)?
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1answer
2k views

Effect of 260/230 values on PCR

To what extent does the 260/230 value affect the efficiency of PCR reactions ? Do the constituents leading to a low 260/230 value (EDTA, guanidine salts and oligosaccharides) inhibit PCR ? I have been ...
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584 views

Origin of the 260/280 ratio?

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, ...
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10k views

What are the roles of guanidine-HCl and ethanol in binding of DNA to silica?

I'm trying to understand how exactly the binding to silica gel (in kits) step works and I cannot find any papers which provide an explanation of the physics or chemistry; especially on the way that ...
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Which kit provides the highest yield and purity of extracted DNA from FFPE tissue samples?

I'm looking to extract DNA for sequencing from FFPE (Formalin fixed paraffin-embedded) tissue samples. I'd like to use a commercial kit that provides a high yield of good quality DNA.
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1answer
21k views

NaCl role in CTAB - DNA complex in DNA extraction

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 ...
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1answer
9k views

Isopropanol precipitation of DNA - duration and magnitude of cold storage

DNA prep protocols often include a final precipitation step with alcohol, often isopropanol, where the DNA must be kept in the alcohol, at a low temperature such as -20C or -70C, often overnight. ...
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1answer
3k views

DNA content in plant seeds vs. fruit flesh

Is there a publication comparing DNA yield and/or PCR-amplifiability after extraction from fruit flesh (like apples, oranges, cherries etc) in comparison to seeds of the same fruits? I would prefer a ...
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1answer
104 views

Clarification of the procedures to amplify cDNA needed

The typical procedure to obtain cDNA from genomic extracted DNA is the following: RNA is extracted from desired tissue, RNA/DNA hybrid is obtained by virtue of reverse transcriptase RNA dependent DNA ...
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2k views

Why is proteinase K digestion performed at 50 °C?

Many DNA isolation protocols use a Proteinase K digestion to remove proteins. This is often performed at 50 °C. Why is this?
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8k views

How to DIY preserve pet DNA today so that it can be used in 20 years

I know that there are companies that offer a kit+storage of your pet DNA for around $2k. My question is if there is any other option for doing this yourself with a thought in mind that in 20+ years ...