Category Archives: Cloning

Please know that there is only one manufacturer of the authentic CEM3340.

The widow of analog synth chip pioneer Doug Curtis has spoken out against the practice of cloning vintage music technology, saying that she is deeply saddened by the attempt of others to trade on [Curtiss] name.

Mary Curtis didnt say which company she was referring to by name, but budget gear manufacturer Behringer recently confirmed it wasmaking replicas of the CEM3340 chip that featured in the original Oberheim OB-Xa synth.

While Behringer is legally allowed to make replicas of theCEM3340 because the patents have expired, Curtis Electromusic still exists, and still makes its ownchips for analog synth gear.

Many of you who are active on synth forums have recently contacted us regarding another companys claim of producing VCO chips that are the equivalent to the CEM3340 that was used in many legendary synthesizers, Curtis said in a statement on Facebook.

To avoid any confusion, please know that there is only one manufacturer of the authentic CEM3340 designed by my late husband, Doug Curtis. Any claims, use of this product designation, and use of the name Curtis Electromusic by other companies are made without permission from OnChip Systems (our current company name) or the Curtis Family.

As much as Doug would be humbled and so very happy about the legacy his products enjoy, we can assure you that as a person of the highest integrity he would be deeply saddened by the attempt of others to trade on his name and to make unsubstantiated claims of equivalency to his original inventions.

Benringers parent company Music Group has been making chips through its Coolaudio subsidiaryfor 17 years, and as Uli Behringer has noted, itsproducts have also been used by Dave Smith Instruments and Elektron to make their own gear.

The general rule and the law clearly describe that technology is free for everyone to use, provided it is not protected, Behringersaid in defense of synth cloning earlier this month. You may have a different personal view, but thats how our society and every industry works again why the law has been designed the way it is.

Behringer has said that he wants to clone the Minimoog, ARP 2600 and OSCar, with a replica Oberheim OB-Xa also looking likely. However, it could be a while before they come to market the companys recentDeepMind 12 synth took over three years to develop. [via Synthtopia]

Read next: 8 classic synths that are crying out to be reissued

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Curtis chip company speaks out against cloning vintage synth technology - FACT

How can a legally binding agreement on human cloning be established? Science Daily Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. In an attempt to address the issue, the UN formulated a Declaration on Human ... and more »

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How can a legally binding agreement on human cloning be established? - Science Daily

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Apple announced a new video editing app called Clips in a company blog post Tuesday. Clips allows users to overlay augmented reality filters, speech bubbles, emojis and even music onto pictures and video in a manner similar to Snapchat.

Gene Munster, a veteran Apple analyst, suggested to CNBC's "Squawk Alley" program that Clips is not a Snapchat competitor, however, as the app doesn't have a built-in social network. Instead, Clips videos and images are intended to be shared on other platforms like Instagram, Facebook or through Apple's own Messages feature. The app automatically recommends shares based on who appears in videos or who the user shares content with most frequently.

AR certainly appears to have caught Apples eye of late, with CEO Tim Cook suggesting that the technology could be as revolutionary as the iPhone was when it first launched. Industry analysis backs up that prediction: In December, the International Data Corporation (IDC) forecast that shipments of AR-ready headsets will reach 15 million over the next three years.

"AR may just be on track to create a shift in computing significant enough to rival the smartphone," Jitesh Ubrani, senior research analyst for IDC Mobile Device Trackers, said in a statement at the time. "However, the technology is still in its infancy and has a long runway ahead before reaching mass adoption."

In the "Squawk" interview, Munster similarly suggested that "the future of computing" will shift focus to AR; the program also addressed that Apple is rumored to be working on some sort of glasses wearable that might compete against Snap's own Spectacles hardware.

Snap is likely sweating the proliferation of copycat apps that continue to pop up following its initial public offering of stock at the beginning of the month. While some competitor platforms like InstagramStories have appeared to take a big bite out of Snapchat'suser base, Clips might arrive as less of a direct threat though still an app to keep a close watch on, as AR is an area that makes sense for Snap to invest innow that it's more flush with cash for research and development.

For marketers,AR applications are fairly rudimentary at the moment, with the most popular use cases found in Snapchat'sSponsored Lenses or games that can tie to real-world locations like Pokemon Go. However, as the technology evolves, it has the potential to revolutionize sectors like retail, allowing consumers to scan and visualize products both in-store and at home.

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Apple joins Snapchat cloning craze with Clips - Marketing Dive

PUNE: A judge from a small cause court in Pune recently fell victim to debit card cloning in which fraudsters made away with Rs 47,930. The judge, S J Gharat, registered a complaint with the Deccan Gymkhana police station on Sunday.

Gharat had received an SMS on February 20 about Rs 50 being debited from her salary account at Axis Bank's FC Road branch. The money was deposited back immediately. Minutes later, Rs 1,600 was debited from the account, but she ignored the SMS thinking that the money would be re-deposited, police said.

The fraudsters withdrew more money several times till February 27, but Gharat did not receive most of the SMSs of the transactions. She saved one of them and obtained a statement of the account from an ATM. The printout indicated that 19 transactions had been carried out even though she had not used her debit card or shared her PIN or any other card verification information.

Gharat quickly blocked her card the same day, and filed a complaint with the bank. She sought an inquiry and told the bank to deposit the money in her account as she was not at fault.

"The bank has assured me that they will refund the amount, but the money has not been deposited in my account. I was directed to register an FIR. I have filed a complaint against unidentified men with Shivajinagar police station," Gharat told TOI on Monday.

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It looks as if Behringer has another vintage synth in its sights.

TomOberheims OB-Xa synth from 1980 could be the next instrumentto be cloned by budget gear company Behringer.

As Synth Anatomy notes, Behringers parent company Music Group applied for a trademark for OB-Xa last year, and is in the process of making replicas of theCurtis CEM3340 chip that featured in the original OB-Xa.

In 2000, company founder Uli Behringer acquired a chip manufacturer called Coolaudio, which has been tasked with creating replicas of old technology something thats legal under patent law. All patents expire after around 20 years and hence the 40 year old Curtis and SSM technology is now public domain, Behringer said in a recent forum post.

According to Behringer, the first batch of these replica chips has already been received. It is a 100% exact replica of the Curtis CEM3340 which even includes the 40 year old, 8 micron manufacturing process, he said.

We are continuing to invest in reviving other legacy Curtis and SSM semiconductors which will allow us to bring back classic synths all in the most authentic way. We have also reissued the 3320 filter chip which should be arriving in a few months.

Behringer also arguedthat companies such as Dave Smith and Elektron currently use Coolaudio chips, saying that theyobviously have no concerns with cloning in general and with using our chips in their products.

Behringer hasnt confirmed the existence of a new OB-Xa, but it seems likely. In recent weekshe has said that the companyis planning a whole range of affordable analog synths, as well as clones of the Minimoog, OSCar and ARP 2600.

As well as planning a raft of clones, Behringeris making brand new synths based on its own designs, including the 12-voice DeepMind 12.

Read next: 8 classic synths that are crying out to be reissued

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Behringer could be cloning the classic Oberheim OB-Xa synth - FACT

TA cloning is a subcloning technique that avoids the use of restriction enzymes[1] and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together. PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3' end of the product. Such PCR amplified inserts are cloned into linearized vectors that have complementary 3' thymine overhangs.[2]

The insert is created by PCR using Taq DNA polymerase. This polymerase lacks 3' to 5' proofreading activity and, with a high probability, adds a single, 3'-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanines at the 5' end as this maximizes probability of Taq DNA polymerase adding the terminal adenosine overhang.[3] Thermostable polymerases containing extensive 3 to 5 exonuclease activity should not be used as they do not leave the 3 adenine-overhangs.[4]

The target vector is linearized and cut with a blunt-end restriction enzyme. This vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. It is important to use ddTTP to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end.[5] Manufacturers commonly sell TA Cloning "kits" with a wide range of prepared vectors that have already been linearized and tagged with an overhanging thymine .

Given that there is no need for restriction enzymes other than for generating the linearized vector, the procedure is much simpler and faster than traditional subcloning. There is also no need to add restriction sites when designing primers and thus shorter primers can be used saving time and money. In addition, in instances where there are no viable restriction sites that can be used for traditional cloning, TA cloning is often used as an alternative. The major downside of TA cloning is that directional cloning is not possible, so the gene has a 50% chance of getting cloned in the reverse direction.[1]

TOPO cloning

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TA cloning - Wikipedia

John Heubusch is the classic "local boy makes good" story.

A House aide in the 1980s who later was the Labor Department's chief of staff during former President George H.W. Bush's administration, he went on to work for Gateway Computers, ran owner Ted Waitt's foundation and is now the executive director of the Ronald Reagan Presidential Foundation and Institute.

His next job: tackling the Second Coming.

Heubusch is the author of the just-released novel The Shroud Conspiracy, dubbed a DaVinci Code meets Indiana Jones thriller that considers the cloning of Jesus. It reads so well that a sequel has been ordered by Simon & Schuster imprint Howard Books, and Heubusch is in talks for a movie.

While he tapped his Washington roots researching the book, it is not a D.C. whodunit.

"When I tell people I've written a book and its sequel, to a person they all expect it's a Brad Meltzer-type novel given all my political experience, the Hill, the administration, lobbying. That's their expectation. But the subject matter I've chosen is far, far away from the halls of Congress," he said.

However, it does touch on a subject that is in the headlines. "One of the fundamentally important elements in the book is all about human cloning," Heubusch said. "From a research nugget standpoint, it's interesting to know that while human cloning seems to be outrageous, it's actually not outlawed in the U.S. There's no federal law prohibiting it."

At a book party for the Shroud Conspiracy a block from the White House Thursday evening, the author said that he has thought about the subject since he was 17, but added, "I don't really know what if feels like to be an author, but book was published just 48 hours ago."

Still, he added, it was already No. 30 on Amazon's best seller list.

Also from the Washington Examiner

"Shut your mouth," one audience member yelled.

03/19/17 3:35 PM

Paul Bedard, the Washington Examiner's "Washington Secrets" columnist, can be contacted at pbedard@washingtonexaminer.com

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Reagan library director taps bigger subject: Cloning Jesus - Washington Examiner

Calico cats are domestic cats with a spotted or particolored coat that is predominantly white, with patches of two other colors (often, the two other colors are orange and black). Outside North America, the pattern is more usually called tortoiseshell-and-white. In the province of Quebec, Canada, they are sometimes called chatte d'Espagne (French for '(female) cat of Spain'). Other names include brindle, tricolor cat, tobi mi-ke (Japanese for 'triple fur'), and lapjeskat (Dutch for 'patches cat'); calicoes with diluted coloration have been called calimanco or clouded tiger. Occasionally, the tri-color calico coloration is combined with a tabby patterning. This calico patched tabby is called a caliby.[1]

"Calico" refers only to a color pattern on the fur, not to a breed.[2] Among the breeds whose standards allow calico coloration are the Manx, American Shorthair, British Shorthair, Persian, Japanese Bobtail, Exotic Shorthair, Siberian, Turkish Van, Turkish Angora and Norwegian Forest Cat.

This condition arises when the individual cat has 2-X chromosomes. This is most common with females. However, rarely, a male cat is conceived with 2-X chromosomes (in addition to its Y chromosome). Because genetic determination of coat colors in calico cats is linked to the X chromosome, calicoes are nearly always female, with one color linked to the maternal X chromosome and a second color linked to the paternal X chromosome.[2][3] Because males only have one X chromosome, a male calico would have to have a rare condition where they have three sex chromosomes (two X chromosomes and one Y chromosome) in order to be calico. In addition to other symptoms caused by the condition, these male calicos are often sterile.

There is also a calico cat referred to as a Dilute Calico. Dilute Calicos are not necessarily rare. They are recognized by their grey, silver, and gold colors instead of the traditional white, black, brown or red patched coat of a calico. Dilute calicos are also called light calicos; because they usually have no dark colored fur.

The coat pattern of calico cats does not define any breed, but occurs incidentally in cats that express a range of color patterns; accordingly the effect has no definitive historical background. However, the existence of patches in calico cats was traced to a certain degree by Neil Todd in a study determining the migration of domesticated cats along trade routes in Europe and Northern Africa.[4] The proportion of cats having the orange mutant gene found in calicoes was traced to the port cities along the Mediterranean in Greece, France, Spain and Italy, originating from Egypt.[5]

In genetic terms, calico cats are tortoiseshells in every way, except that in addition they express a white spotting gene. There is however one anomaly: as a rule of thumb the larger the areas of white, the fewer and larger the patches of ginger and dark or tabby coat.[citation needed] In contrast a non-white-spotted tortoiseshell usually has small patches of color or even something like a salt-and-pepper sprinkling. This reflects the genetic effects on relative speeds of migration of melanocytes and X-inactivation in the embryo.[6]

Serious study of calico cats seems to have begun about 1948 when Murray Barr and his graduate student E.G. Bertram noticed dark, drumstick-shaped masses inside the nuclei of nerve cells of female cats, but not in male cats. These dark masses became known as Barr bodies.[7] In 1959, Japanese cell biologist Susumu Ohno determined the Barr bodies were X chromosomes.[7] In 1961, Mary Lyon proposed the concept of X-inactivation: one of the two X chromosomes inside a female mammal shuts off.[7] She observed this in the coat color patterns in mice.[8]

Calico cats are almost always female because the locus of the gene for the orange/non-orange coloring is on the X chromosome. In the absence of other influences, such as color inhibition that causes white fur, the alleles present in those orange loci determine whether the fur is orange or not. Female cats like all female placental mammals normally have two X chromosomes. In contrast, male placental mammals, including chromosomally stable male cats, have one X and one Y chromosome.[2][7][9] Since the Y chromosome does not have any locus for the orange gene, there is no chance that an XY male could have both orange and non-orange genes together, which is what it takes to create tortoiseshell or calico coloring.[citation needed]

One exception is that in rare cases faulty cell division may leave an extra X chromosome in one of the gametes that produced the male cat. That extra X then is reproduced in each of his cells, a condition referred to as XXY, or Klinefelter syndrome. Such a combination of chromosomes could produce tortoiseshell or calico markings in the male, in the same way as XX chromosomes produce them in the female.[citation needed]

All but about one in three thousand of the rare calico or tortoiseshell male cats are sterile because of the chromosome abnormality, and breeders reject any exceptions for stud purposes because they generally are of poor physical quality and fertility. In any event, because the genetic conditions for calico coloring are X linked, a fertile male calico's coloring would not have any determination in the coloring of any male offspring (who would receive the Y, not the X chromosome from their father).

As Sue Hubble stated in her book Shrinking the Cat: Genetic Engineering before We Knew about Genes,

The mutation that gives male cats a ginger-colored coat and females ginger, tortoiseshell, or calico coats produced a particularly telling map. The orange mutant gene is found only on the X, or female, chromosome. As with humans, female cats have paired sex chromosomes, XX, and male cats have XY sex chromosomes. The female cat, therefore, can have the orange mutant gene on one X chromosome and the gene for a black coat on the other. The piebald gene is on a different chromosome. If expressed, this gene codes for white, or no color, and is dominant over the alleles that code for a certain color (i.e. orange or black), making the white spots on calico cats. If that is the case, those several genes will be expressed in a blotchy coat of the tortoiseshell or calico kind. But the male, with his single X chromosome, has only one of that particular coat-color gene: he can be not-ginger or he can be ginger (although some modifier genes can add a bit of white here and there), but unless he has a chromosomal abnormality he cannot be a calico cat.[5]

It is currently impossible to reproduce the fur patterns of calico cats by cloning. Penelope Tsernoglou wrote "This is due to an effect called x-linked inactivation which involves the random inactivation of one of the X chromosomes. Since all female mammals have two X chromosomes, one might wonder if this phenomenon could have a more widespread impact on cloning in the future."[10]

Calico cats may have already provided findings relating to physiological differences between male and female mammals. This insight may be one day broadened to the fields of psychology, psychiatry, sociology, biology and medicine as more information becomes available regarding the complete effect of random X-inactivation in female mammals.[7][9][11]

Cats of this coloration are believed to bring good luck in the folklore of many cultures.[12] In the United States, these are sometimes referred to as money cats.[13] A cat of the calico coloration is also the state cat of Maryland in the United States.[14] In the late nineteenth century, Eugene Field published "The Duel", a beloved poem for children also known as "The Gingham Dog and the Calico Cat." In Japan, the Maneki-Neko figures depict Calico cats, bringing good luck.

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Calico cat - Wikipedia

New York Times

Cloning Yo-Yo Ma: This Week's 8 Best Classical Moments New York Times In addition to reviews, features and news during the week, our critics and reporters collect the best of what they've heard: notes that sent shivers down their spines, memorable voices, quotations that cut to the heart of the story. Read the rest of ... and more »

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Cloning Yo-Yo Ma: This Week's 8 Best Classical Moments - New York Times

Semi-automated Tip Snip cloning of restriction fragments into and out of plasmid polylinkers

Emory University School of Medicine, Department of Biochemistry, O. Wayne Rollins Research Center, Atlanta, GA

BioTechniques, Vol. 62, No. 3, March 2017, pp. 99106

Supplementary Material

Abstract

Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps. Cohesive ends of unwanted restriction fragments are snipped off by additional restriction endonucleases. The resulting short fragments (snippets) are eliminated by hybridization to complementary oligonucleotides (anti-snippets) and subsequent size-selection spin-column chromatography. Unwanted linear donor vectors are ligated to double-stranded oligonucleotides (unlinkers) so that only the desired insert and recipient plasmid form circular DNA capable of transforming bacteria. This new method is compatible with high-throughput processing and automated liquid handling, and because no specialized vectors, reagents, selection schemes, or analytical techniques are required, the barriers to adoption are low.

DNA synthesis costs are decreasing (1), but the assembly and cloning of synthetic DNA remains relatively labor-intensive and expensive. Nucleoside phosphoramidites are chemically synthesized on large scales. Single-stranded oligonucleotides and double-stranded synthetic genes are custom manufactured by machines, so turnover is rapid, throughput is high, and production costs are relatively low. Automation and miniaturization have decreased the per-unit cost of synthesizing gene-length (2 kb) DNAs, but further innovations are required to overcome practical limitations in length, nucleotide composition, accuracy, and yield (1). It is not yet feasible to have every new construct synthesized with its vector de novo, so PCR products and synthetic genes are most often cloned into plasmids and then sequenced.

Cloned parts are often assembled into larger constructs by subcloning, but this classical approach is recalcitrant to automation for three reasons. First, robots that can load agarose gels and purify particular restriction fragments have not yet been invented. Furthermore, DNA purification, restriction digests, and ligation reactions arent reliably efficient, making monitoring and troubleshooting necessary. Finally, the design of cloning experiments is idiosyncratic, so the development of software algorithms that emulate decision making by experienced molecular biologists is non-trivial. The per-unit labor cost of subcloning (~10 h of labor per attempt, not including incubation times) far exceeds those of reagents (e.g., enzymes and purification kits).

METHOD SUMMARY

Tip Snip cloning uses restriction enzymes to shorten unwanted DNA fragments; the unwanted sticky ends are then neutralized by synthetic oligonucleotides. By eliminating the need to gel purify the desired restriction fragments, Tip Snip enables automation of the entire subcloning workflow.

The high cost of molecular cloning has motivated the invention of new methods (2-5). In general, one-pot sequence-specific recombination reactions, such as those catalyzed by recombinases (e.g., Gateway cloning) (6, 7), thermostable polymerases (overlap extension PCR) (8), thermostable ligases (ligase chain reaction) (9), or combinations of exonuclease, polymerase, and ligase (Gibson assembly or ligase-independent cloning (10,11) are the most amenable to high-throughput and automated techniques (12). These protocols are less labor-intensive than traditional cloning workflows with discrete steps but are more difficult to troubleshoot. Another drawback of many seamless assembly techniques is their reliance on PCR or gene synthesis to create fragments with compatible ends. Every part must be re-sequenced each time it is seamlessly combined with another element and re-cloned (Special News Report. Weaver, J. 2015. BioTechniques. 59:II-III.), because the DNA polymerase I homologs used in PCR are three to five orders of magnitude less accurate than those responsible for in vivo plasmid replication and repair. Next-generation sequencing techniques lower per-unit cost, but they cannot be applied to individual plasmids. Thus, many synthetic biologists continue to assemble parts by manual subcloning.

I therefore sought a way to automate the conventional restriction endonuclease/T4 DNA ligasedependent subcloning workflow. Golden Gate assembly, which utilizes type IIS restriction endonucleases (13), and 2ab assembly (14), which utilizes plasmids with two selectable markers separated by a unique restriction site, obviate gel purification but necessitate the employment of specialized vectors incompatible with those of other cloning standards. The three antibiotic assembly (3A) protocol (15) was specifically designed to assemble parts compatible with the seminal BioBrick Assembly Standard (RFC10) used by many synthetic biologists (16). Two donor plasmids carrying parts and a recipient plasmid encoding a counter-selection marker along with a selectable marker different than those of the donors are digested with different pairs of restriction enzymes. All six of the resulting restriction fragments are ligated together and used to transform Escherichia coli. The desired recombinant construct is distinguished from the parental plasmids using an antibiotic and the counter-selection scheme. This technique circumvents gel purification, but sacrifices efficiency for convenience. Three-fragment ligations dont occur as frequently as two-fragment reactions, particularly when three other unwanted fragments with compatible cohesive ends are present. The extraneous DNA also inhibits heat shock transformation of chemically competent E. coli (17), and electroporation is sensitive to salts in ligation reactions so it is less amenable to high-throughput experiments. Here, I describe a set of expedients that in combination facilitate efficient and reliable cloning of DNA into or out of almost any existing plasmid polylinker (multiple cloning site) without the need for PCR amplification or gel purification. Materials and methods

The approach described here builds upon the following classical cloning techniques (18), except as noted. A more detailed step-by-step protocol is included in the Supplementary Material. Plasmids were purified from transformed E. coli using silica spin columns (QIAGEN, Valencia, CA) and hydrated Sephadex G-50 (GE Healthcare Life Sciences, Pittsburgh, PA) in empty spin columns (Epoch Life Science, Missouri City, TX) as directed by their manufacturers. Restriction digests were set up as recommended by the supplier [New England BioLabs (NEB), Ipswich, MA]. Whenever possible 2 g of DNA (6 nM for a 5 kb plasmid) were digested to completion (or nearly so) at 37C overnight with 2040 U of each restriction enzyme (12 nM) in 100 L total reaction volume. Approximately 20 fmol of digested, purified recipient plasmid and 20 fmol of digested insert (and donor plasmid) were reacted with 0.31 Weiss units of T4 DNA ligase in NEB T4 DNA ligase buffer containing 5% polyethylene glycol (molecular weight: 8000) (19). The reactions were temperature cycled in a Bio-Rad (Hercules, CA) MJ mini thermocycler between 10C for 30 s and 30C for 30 s for a total of 418 h (20).

Chemically competent E. coli OmniMax2 cells (Thermo Fisher Scientific, Waltham, MA) were prepared according to Inoue et al. (17). For each transformation, up to 1.25 ng total DNA in ligation reactions were used to transform 25 L of competent cells in the thermocycler. The transformants were spread on lysogeny broth medium (LB) agar plates containing 100 g/mL ampicillin. Some agar plates also contained inducer and a histochemical substrate as described below. Some colonies were adsorbed to a nitrocellulose filter and transferred colony-side up to fresh LB-ampicillin plates supplemented with inducer (1 mM IPTG, 10 g/mL tetracycline, 0.4% L-arabinose or 0.4% rhamnose) and 2 mg/25 mL plate X-gal (for colonies carrying lacZ expression vectors). Additional information the reagents and materials used to culture the bacteria can be found in the Supplementary Material. Results and discussion

The Tip Snip cloning workflow begins with plasmids prepared via alkaline lysis and silica spin-column chromatography. A QIAcube robotic workstation (QIAGEN) can be used to automate this and other purification protocols. To eliminate small molecules that might inhibit restriction endonucleases or broaden their sequence specificity (21), plasmids are further purified via manual gel-filtration spin-column chromatography. Recipient and donor plasmids are digested as usual with restriction enzymes that produce fragments with compatible cohesive ends. Additional restriction enzymes recognizing sites in the polylinker are used to shorten undesired restriction fragments (the stuffer fragment of the recipient plasmid or the donor plasmid of a subcloning experiment) (Figure 1).

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