Isolating DNA fragments

Materials:

0.8 % agarose gel in 1x TAE

Digested DNA

Glass Milk

NaI solution

New Wash

Procedure:

1) Run digested DNA out on agarose gel slowly (70 V on BioRad gel)

2) Use long wave UV lamp to visualize bands. Cut out band with scalpel. Cut smallest possible piece.

3) Put gel slice in an eppendorf tube and weigh to figure out volume of gel slice. (empty tube approx. 1 g).

4) Add 3 vol of NaI solution (gel slice is usually ~200 mg, therefore, add 600 ul NaI).

5) Melt gel slice in 55 degree water bath.

6) Add 5 ul glass milk and mix (vortex glass milk vigorously to resuspend before adding to DNA mx). Incubate on ice ~5 min.

7) Spin down glass milk quickly. Decant. Add 1 ml New Wash and vortex. Do a total of 3 washes.

8) Spin down hard to pellet glass milk. Aspirate off liquid and air dry.

9) Resuspend pellet in 30-50 ul TE pH 7.5. Heat at 55degrees for 5 min.

10) Spin down glass milk and save supernatant. This is your DNA.

NaI solution

Mix 90.8 g NaI and 1.5 g Na2SO3 (note: sulfite!) to a final volume of 100 ml (cover with foil while stirring, light sensitive). Filter through #1 Whatman paper add 0.5 g Na2SO3. Store in dark at 4degreesC.

New Wash for 500 ml

50% EtOH 250 ml

0.1 M NaCl 2.92 g

10 mM Tris 7.5 5 ml 1M

1 mM EDTA 1 ml 0.5 M

E. coli Transformation

Long

1) Get 200ul aliquots of E. coli (DH5a for normal transformation or DE3 for expression) from -80C freezer and let thaw on ice.

2) Add DNA

For plasmid: 1ul DNA desired

For ligation: 10ul ligation reaction

-Also include a negative control with no DNA.

3) Incubate for 30min on ice.

4) Heat shock for 90sec at 42C.

5) Incubate for 1min on ice.

6) Add 1ml LB.

7) Agitate at 37C for 45min – 2hrs (1hr is good).

8) Spin down and pour off LB leaving ~100ul.

9) Resuspend cells in the ~100ul.

10) Plate the entire suspension on appropriate prewarmed plates.

11) Incubate at 37C overnight.

Short

Note: Use for amplifying AmpR plasmids only.

1) Get 200ul aliquots of E. coli (DH5a) from -80C freezer and let thaw on ice.

2) Aliquot 50ul of cells into tubes.

3) Add 1ul DNA desired.

4) Incubate on ice 5min-30min.

5) Plate on prewarmed LB-Amp plates. (Plate the whole thing--no spinning, no nothing).

6) Incubate at 37C overnight.

Blue/White Selection

-Used for plasmids like Bluescript.

-No insert=Blue. Insert=White.

-Do long transformation of ligations basically as described above.

-While cells are agitating,

A) Spread X-Gal and IPTG on plates.

X-Gal (50mg/ml): 20ul

IPTG (1M): 10ul

B) Incubate at 37C to dry (about 30min).

E. coli Miniprep

1) Grow 2ml culture o/n.

2) Spin down. Pour off all but ~100ul of sup. Resuspend cells in the ~100ul.

3) Add 130ul P1. Resuspend.

4) Add 130ul P2. Mix immediately.

5) Add 182ul N3. Mix immediately.

6) Spin 10 min at 4C. (Removes genomic DNA and debris).

7) Transfer sup to a fresh eppendorf.

8) Add 900ul ice-cold 100% EtOH (bottom shelf in door of -20C freezer). Mix thoroughly.

9) Spin 15min at 4C. Discard sup.

10) Wash pellet once with ~400ul ice-cold 70% EtOH.

11) Dry pellet on rack or in speedy-vac.

12) Resuspend in 50ul water. Use 3-10ul for digest.

Molecular Biology Protocols Online

One-Tube Isolation of DNA from Mouse Tails

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot093534

Abstract

   

Full Text

   

Full Text (PDF)

Buffers

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.top098210

Abstract

   

Full Text

   

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Caring for Escherichia coli

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot101337

Abstract

   

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Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts

Christopher Jenness, 

David J. Wynne, 

and Hironori Funabiki

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot097113

Abstract

   

Full Text (PDF)

Analysis of Mitotic Checkpoint Function in Xenopus Egg Extracts

Yinghui Mao

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot099853

Abstract

   

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Transformation of Schizosaccharomyces pombe in a 96-Well Format

Assen Roguev, 

Jiewei Xu, 

and Nevan J. Krogan

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot091942

Abstract

   

Full Text

   

Full Text (PDF)

DNA Preparation from Schizosaccharomyces pombe

Assen Roguev, 

Jiewei Xu, 

and Nevan J. Krogan

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot091959

Abstract

   

Full Text

   

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Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Maxipreps

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot093351

Abstract

   

Full Text

   

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A Single-Step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot093500

Abstract

   

Full Text

   

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Gateway Recombinational Cloning

John S. Reece-Hoyes

   

and Albertha J.M. Walhout

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.top094912

Abstract

   

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Propagating Gateway Vectors

John S. Reece-Hoyes

   

and Albertha J.M. Walhout

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot094920

Abstract

   

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Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone

John S. Reece-Hoyes

   

and Albertha J.M. Walhout

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot094938

Abstract

   

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Using Multisite LR Cloning to Generate a Destination Clone

John S. Reece-Hoyes

   

and Albertha J.M. Walhout

Cold Spring Harb Protoc 2018; doi:10.1101/pdb.prot094946

Abstract

   

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Preparation of DNA from Embryonic Stem Cells or Other Cultured Cells

Richard Behringer, 

Marina Gertsenstein, 

Kristina Vintersten Nagy, 

and Andras Nagy

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot092718

Abstract

   

Full Text

   

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Analysis of DNA Methylation in Mammalian Cells

Paul M. Lizardi, 

Qin Yan, 

and Narendra Wajapeyee

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.top094821

Abstract

   

Full Text

   

Full Text (PDF)

Methylation-Specific Polymerase Chain Reaction (PCR) for Gene-Specific DNA Methylation Detection

Paul M. Lizardi, 

Qin Yan, 

and Narendra Wajapeyee

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094847

Abstract

   

Full Text

   

Full Text (PDF)

Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis

Paul M. Lizardi, 

Qin Yan, 

and Narendra Wajapeyee

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094854

Abstract

   

Full Text

   

Full Text (PDF)

High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation

Paul M. Lizardi, 

Qin Yan, 

and Narendra Wajapeyee

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot094862

Abstract

   

Full Text

   

Full Text (PDF)

Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot093419

Abstract

   

Full Text

   

Full Text (PDF)

Growing Bacteriophage M13 in Liquid Culture

Michael R. Green

   

and Joseph Sambrook

Cold Spring Harb Protoc 2017; doi:10.1101/pdb.prot093435

Abstract

   

Full Text

   

Full Text (PDF)

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Tailed spider trapped in 100-million-year old amber offers insight in evolution

A spider-like creature bearing a long tail has been discovered in an amber from Myanmar dating back about 100 million years.

"There's been a lot of amber being produced from northern Myanmar and its interest stepped up about ten years ago when it was discovered this amber was mid-Cretaceous," said Paul Selden, a palaeontologist at the University of Kansas and an author of a paper on the ancient arachnid.

"Therefore, all the insects found in it were much older than first thought," Selden told the university in an interview.

The finding is published Monday in a paper in Nature Ecology & Evolution by an international team of researchers from the United States, China, Germany and the United Kingdom.

According to the paper, the creature shares many similarities with modern spiders including fangs, eight legs and silk-producing spinners.

Moreover, it also bears a long flagellum, or a tail, at its rear, which is not commonly seen among modern spiders.

Thought the ancient arachnid has a tiny body, only about 2.5 millimeters long, it had a tail nearly twice of its body length.

"Any sort of flagelliform appendage tends to be like an antenna," Selden said, "It's for sensing the environment. Animals that have a long whippy tail tend to have it for sensory purposes."

The tailed arachnid, named Chimerarachne after the Greek mythological Chimera, documents a key transition stage in spider evolution.

Previously, Selden and his colleagues had found similar insects without spinnerets from the much older Devonian and Permian periods.

"That's why the new one is really interesting, apart from the fact that it's much younger -- it seems to be an intermediate form," Selden said.

"In our analysis, it comes out sort of in between the older one that hadn't developed the spinneret and modern spider that has lost the tail," he added.

Cloned monkeys could help development of drugs for human brain diseases

Chinese scientists' successful cloning of monkeys could eventually help the development of new drugs to treat human diseases, like brain and nervous system problems, a leading Chinese neuroscientist said.

At the end of 2017, a non-human primate research facility under the Beijing-based Chinese Academy of Sciences (CAS) produced two cloned macaques.

Allaying fears that cloning monkeys for research could ultimately lead to human cloning, Poo Muming, director of the Institute of Neuroscience at CAS, told Xinhua, "We have no intention to clone humans."

The macaque cloning was done for a humane reason, he said: "Because this is the species that will really help human health and cure human disease."

The cloning will reduce the use of large numbers of primates for research, especially in the West.

In 2016, the United States used over 71,000 primates for research or experimentation, according to the U.S. Department of Agriculture's animal and plant health inspection service.

U.S. pharmaceutical companies import at least 50,000 monkeys every year to test the efficacy of different drugs and determine safe doses for human clinical trials, Poo said.

"That is a huge number of monkeys used, which you should consider unethical," he said.

Since these monkeys have diverse genetic backgrounds, researchers must use a large number to make sure that the observed effects are due to treatment and not because of the genetic variation.

"Cloning is really the way to go," Poo said. "Because it reduces the interference (of) the diversity of genetic background on drug development. So it is for (that) ethical reason that we (have cloned) monkeys."

Darren Griffin, genetics professor at the University of Kent, holds the same view.

"If they can produce these cloned animals that means that you could use fewer animals for ... research, rather than the number being used at the moment," he said in a previous interview with Xinhua.

At present, the medical community mostly uses mice as a model to research cures for human diseases. But for many diseases, especially brain diseases, drugs that are developed using mice as model have failed clinical trials in humans, Poo said.

"Basically all big pharmas have given up developing neuro drugs," he said, citing two decades of drug development failure, with hundreds of billions of dollars spent on each drug.

"Next, we want to develop clones of animals that carry brain disorders," he said, especially degenerative or development diseases that have clear genetic causes.

Poo said he would like to focus on Alzheimer's, Parkinson's, autism and ALS, a nervous system disorder that causes disability.

Regarding the debate on the ethics of non-human primate cloning, he said every new technology is a two-edged sword: "There will be discussions and we'll see how the society deals with it."

In the 1970s, when genetic engineering technology first came out, he said everybody was worried about its harmful effect on humans.

"But so far, it has been 40 to 50 years and you don't see any real problem yet," he said.

"Cloned monkeys could be very valuable in studying specific aspects of human disease," said Tom Holder, director of Speaking of Research, an international organization that supports the use of animals in scientific labs.

"The breakthrough has shown that it is possible," Holder said in an emailed interview to Xinhua. "But it is too early to make any conclusions on whether the method will be able to be repeated efficiently and reliably."

Holder also said non-human primate research is subject to increased ethical and welfare considerations and is generally used only when other species are unsuitable for the research.

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google theme: 175 USD

language translator： 99 USD

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