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

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47k views

Why do we add salt when precipitating DNA?

All the DNA extraction protocols I have seen involve adding salts to the extraction buffer. What is the purpose of the salts? What happens if they aren't included?
11
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1answer
3k 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 ...
9
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2answers
1k views

What is causing my problem with very low yields when isolating a 42kb yeast plasmid?

I have to isolate a large plasmid from yeast and transform it in E. coli. After transformation, I often get no colonies. One reason for that is the yeast mini prep hasn't worked or the DNA ...
7
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2answers
1k views

How do I prepare and clone from E. coli DNA?

I'm looking for a protocol to get genomic DNA from an E. coli sample so that I can clone a small portion of it using PCR into a plasmid. (< 500 bp in this case). It seems OWW (Open Wet Ware) ...
7
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1answer
550 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 ...
6
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1answer
306 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 ...
5
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4answers
968 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 ...
4
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5answers
768 views

What is a good miniprep protocol for the class room?

I'm trying to find a good protocol for plasmid minipreps and I'm looking at 3 preps I've found: Using phenol/chloroform extract with phenol:chloroform:isoamylalcohol, isopropanol precipitation, 12,...
4
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2answers
110 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 ...
4
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1answer
90 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 ...
4
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1answer
319 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?
4
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2answers
961 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?
4
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1answer
76 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 ...
4
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1answer
99 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)?
4
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1answer
10k 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 ...
4
votes
1answer
727 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 ...
4
votes
1answer
54 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?
3
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0answers
292 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 ...
2
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1answer
412 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 ...
2
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1answer
122 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 ...
2
votes
1answer
4k 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. ...
2
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2answers
42 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 ...
2
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1answer
38 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 ...
2
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1answer
633 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 ...
2
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0answers
204 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, ...
2
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0answers
2k 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 ...
1
vote
1answer
155 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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2answers
376 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 ...
1
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1answer
66 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.
1
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1answer
70 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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0answers
62 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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0answers
35 views

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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0answers
36 views

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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0answers
22 views

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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20 views

plant total DNA electrophoresis

Why does plant total DNA migrate as a single band in agarose gel after electrophoresis, and what kinds of DNA (single, circular, supercoiled, ...) are present in total DNA preparation? Thank you
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0answers
31 views

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 ...
0
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1answer
279 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 ...
0
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0answers
15 views

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 ...
0
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0answers
141 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 ...
0
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0answers
101 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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1answer
2k 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 ...