How was able to make a recombinant DNA?

Making Recombinant DNA

How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:

http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mga/ch10e1.gif


Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated like chemical reagents. Figure 10-1 on the following page gives a general outline of the approach.

The term recombinant DNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and prokaryotes. Recombinant DNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer the alternative name chimeric DNA, after the mythological Greek monster Chimera. Down through the ages, the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different animals. Likewise, recombinant DNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinant DNA technology.
Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 10-2 on page 303. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.top link
Cutting DNA

The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation. Any DNA molecule, from viruses to humans, contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor would they be cut in vivo, because most organisms do not have restriction enzymes.

Let's look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the following sixnucleotide-pair sequence in the DNA of any organism:

more detail : http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mga.section.1549

unescape(), escape()

These are similar to the decodeURI() and encodeURI(), but escape() is used for only portions of a URI.

DBA Checklist Disaster Recovery

1. You must create a disaster recovery plan and include every detail you will need to rebuild your servers.
2. As your SQL Servers change over time, don’t forget to update your disaster recovery plan.
3. Write the disaster recovery plan so that any computer literate person will be able to read and follow it. Do not assume a DBA will be rebuilding the servers.
4. Fully test your disaster recovery plan at least once a year.
5. Re-read all the best practice just mentioned. I’m not kidding. Remember, as DBAs, we are guardians of the organization’s data. This is a huge responsibility.

US Immigration Glossary - Business Nonimmigrant

An alien coming temporarily to the United States to engage in commercial transactions which do not involve gainful employment in the United States, i.e., engaged in international commerce on behalf of a foreign firm, not employed in the U.S. labor market, and receives no salary from U.S. sources.
US Immigration Glossary

10 Jobs With High Pay and Minimal Schooling Required!


1. Air Traffic Controllers: $117,200

These workers make sure airplanes land and take off safely, and they typically top lists of this nature. The median 50% earned between $86,860-142,210, with good benefits. Air traffic controllers are eligible to retire at age 50 with 20 years of service, or after 25 years at any age.

Watching blinking dots on a radar screen that control the lives of hundreds can be stressful, and the job require specialized FAA schooling and on-the-job training. Typically, two to four years of training are needed in order to become fully certified, although previous military experience can cut that time down significantly.

2. Industrial Production Managers: $77,670

They oversee manufacturing activities. A college degree is preferred, but not necessarily mandatory. They often work in industries such as aviation and automobiles.


3. First-Line Police and Detective Supervisors: $69,300

Police officers can advance through the ranks to become supervisors by passing exams and achieving good performance reviews, and advanced training can help win promotions.

4. Funeral Director: $49,620

College programs in mortuary science usually last from two to four years. You typically must also serve a one-year apprenticeship, pass an exam and obtain a state license. Hours can be long and irregular. Dealing with dead bodies and crying relatives isn't for everyone.

5. Police and Sheriff Patrol Officers: $47,460

Police corporals had an average minimum annual base salary of $44,160, according to the International City-County Management Association. But total income can significantly exceed base salary because of overtime pay. And police officers can often retire at half-pay after 25-30 years of service.

Applicants usually must have at least a high school education, and some departments require a year or two of college or even a degree. Rookies are trained at police academies.

6. Advertising Sales Agents: $42,750

20% have a high school degree or less, and 10% have an Associate's degree.

7. Real Estate Brokers and Sales Agents: $39,760

Don't let that figure fool you; the highest 10% earned more than $111,500. While advanced coursework is not necessarily required, new entrants must pass an exam and get a state license. Connections in the community and a willingness to work hard are what really count, but experience and a good housing market also help.

8. Occupational Therapist Assistants: $42,060

These workers usually need an associate degree or a certificate. They work with occupational therapists, helping injured patients recover from, or compensate for, lost motor skills. Job prospects are good in the growing health care field, especially for those with some post-secondary education.

9. Occupational Therapist Aides: $25,000

These employees receive most training on the job. Under supervision of occupational therapists, they also work with injured people. Competition for jobs is tougher for those with only a high school diploma.

10. Physical Therapist Assistants: $41,360

These workers deal with physical therapists, helping patents improve mobility, relieve pain or overcome injuries or disabilities. Those working in home health care services tend to make more on average. Aides, earning an average of $22,000, are trained on the job. Assistants, who have greater responsibilities, typically need an associate's degree.

The Bottom Line

Despite a recession, plenty of career paths can lead to well-paying professions without spending four years or more hitting the books, including opportunities in law enforcement, health care and sales. The goal is to find a job that matches your own particular talents and preferences in addition to supporting your lifestyle.

IT Interview Questions: What is an IP address?

Every device connected to the public Internet is assigned a unique number
known as an Internet Protocol (IP) address. IP addresses consist of four
numbers separated by periods (also called a 'dotted-quad') and look something
like 127.0.0.1. In computer networking, an Internet Protocol (IP) address
consists of a numerical identification (logical address) that network
management assigns to devices participating in a computer network utilizing
the Internet Protocol for communication between its nodes.
[1]
Although
computers store IP addresses as binary numbers, they often display them in
more human-readable notations, such as 192.168.100.1 (for IPv4), and
2001:db8:0:1234:0:567:1:1 (for IPv6). The role of the IP address has been
characterized as follows: "A name indicates what we seek. An address
indicates where it is. A route indicates how to get there."

How to Never Be Nervous Again
_

If even the thought of interviewing makes you nervous, its important to get that emotion under control. The interview is your opportunity to be at your best. If you allow nervousness to control your presentation (or lack thereof), your image may be forever shrouded in the cloud of nervousness that will block the interviewers total view of who you are.

Why do we get nervous? Because of the unknown. We are seeking approval, but we are unsure of ourselves and how we will be perceived. We are afraid we wont get approval, which makes us nervous. And to compound the problem, our increasing nervousness makes it even more difficult to gain that approval, thereby compounding the basis for our fears. Uncontrolled, nervousness can destroy our ability to effectively interview.

But it doesnt have to be that way. The following is a simple technique you can apply to overcome your nervousness in any interviewing situation. It is a technique that I personally use in overcoming my own nervousness, and it will work equally well for you.

One is negative one


Theorem: 1 = -1
Proof:
1 = sqrt(1) = sqrt(-1 * -1) = sqrt(-1) * sqrt(-1) = 1^ = -1

Also one can disprove the axiom that things equal to the same thing are equal to each other.

1 = sqrt(1)
-1 = sqrt(1)
Therefore 1 = -1

As an alternative method for solving:

Theorem: 1 = -1
Proof:
x=1
x^2=x
x^2-1=x-1
(x+1)(x-1)=(x-1)
(x+1)=(x-1)/(x-1)
x+1=1
x=0
0=1
=> 0/0=1/1=1

Check list for Conducting Unit Tests


§ Is the number of input parameters equal to number of arguments?
§ Do parameter and argument attributes match?
§ Do parameter and argument units system match?
§ Is the number of arguments transmitted to called modules equal to number of parameters?
§ Are the attributes of arguments transmitted to called modules equal to attributes of parameters?
§ Is the units system of arguments transmitted to called modules equal to units system of parameters?
§ Are the number of attributes and the order of arguments to built-in functions correct?
§ Are any references to parameters not associated with current point of entry?
§ Have input only arguments altered?
§ Are global variable definitions consistent across modules?
§ Are constraints passed as arguments?
§ When a module performs external I/O, additional interface tests must be conducted.

Again, from Myers:
§ File attributes correct?
§ OPEN/CLOSE statements correct?
§ Format specification matches I/O statement?
§ Buffer size matches record size?
§ Files opened before use?
§ End-of-file conditions handled?
§ I/O errors handled?
§ Any textual errors in output information?
The local data structure for a module is a common source of errors. Test cases should be designed to uncover errors in the following categories:
§ improper or inconsistent typing
§ erroneous initialization or default values
§ incorrect (misspelled or truncated) variable names
§ inconsistent data types
§ underflow, overflow and addressing exceptions
From a strategic point of view, the following questions should be addressed:
§ Has the component interface been fully tested?
§ Have local data structured been exercised at their boundaries?
§ Has the cyclomatic complexity of the module been determined?
§ Have all independent basis paths been tested?
§ Have all loops been tested appropriately?
§ Have data flow paths been tested?
§ Have all error handling paths been tested?

5 worst kinds of used cars to buy

Red Flag #1: No Service Records

Red Flag #2: Vehicle and Accident History Issues

Red Flag #3: Mechanical Problems

A quick look under the car can reveal fluid leaks, but unless you're an expert, you'll need to have someone else do a thorough inspection to make sure it's in good working condition. A PPI, or Pre-Purchase Inspection, is performed by a neutral third party (such as a certified mechanic or automotive technician) and costs about $100 to $200.

Red Flag #4: A Problem Title

Your AutoCheck vehicle report will show any problem titles on the car's record at any point in its history -- everything from hail damage to fire damage. Most of these issues are red flags, but here's just a sampling of those to avoid at all costs: Flood titles, which means the vehicle has received extensive water damage; salvage titles, which mean an insurance company has at some point declared the car a total loss due to sustained damage; and junk titles, which mean the vehicle indicate that a vehicle is not safe enough for use and the car cannot be titled again in that state.

Red Flag #5: A Fraudulent Title

A problem-free title doesn't mean it's valid. Criminals can create counterfeit or fraudulent titles and use them to legally register and then sell stolen cars. If you buy a car without a valid title, it's the worst-case scenario -- you don't legally own the car you just bought.