Lab+6+Molecular+Genetics

== =Bacterial Transformation Lab= =Introduction=

DNA is the genetic material of all living organisms and all organisms use the same genetic code. Genes from one kind of organism can be transcribed and translated when put into another kind of organism. For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use. Human insulin, human growth hormone, and vaccines are produced by bacteria.
 * //Biotechnology//** refers to technology used to manipulate DNA. The procedures are often referred to as **//genetic engineering//**.
 * //Recombinant DNA//** refers to DNA from two different sources. Individuals that receive genes from other species are **//transgenic//**.

Vectors
Vectors are DNA used to transfer genes into a host cell. **//Plasmids//** can be used as vectors to transform bacteria. The host bacterium takes up the plasmid, which includes the foreign gene. Marker genes can be used to determine if the gene has been taken up. Marker genes must have some distinguishable characteristic. For example if you put a gene that enables an ampicillin resistance on the same vector as the same vector as the gene for human insulin production, then any bacteria that grow on an ampicillin plate will be able to produce insulin.

=Materials Needed= The following equipment will be needed for each group:

1 //E. coli// starter plate 4 agar plates: 1 LB plate 2 LB/amp plates 1 LB/amp/ara plate 4 microtubules (ignore the colors of the tubules): 1 microtubule containing transformation solution 1 microtubule containing LB broth 1 empty microtube labeled + (or + DNA) 1 empty microtube labeled - (or - DNA) 1 foam microtube holder 1 container with ice water 1 package of. inoculation loops 1 micropipette and micropipette tips

=Procedure=

Add Transformation Solution (CaCl2)
Use a micropipette to transfer 250 ul of transformation solution (T.S.) to the tube labeled + DNA and another 250 ul to the tube labeled - DNA. Place the tubes back in the foam microtube holder and then float all four of the tubes in a container of ice water for 2 minutes.

Add Bacteria
Use a sterile loop to pick up several colonies of bacteria from the starter plate. This can be done by dragging the loop across the plate so that it lightly scrapes the colonies off the surface. Transfer the bacteria to the + DNA tube by spinning the loop rapidly after it is immersed in the liquid. Repeat this procedure by transferring several colonies of bacteria to the - DNA tube.

Add DNA
Your instructor will provide you with a bottle containing plasmid DNA. Immerse a sterile loop into the bottle containing plasmid DNA. When the center of the loop is coated with a soap-like film, transfer it the + DNA microtube (see photograph below). Use a new sterile loop to transfer a second loopful of plasmid DNA into the same (+ DNA) microtube. The - DNA microtube will not receive any plasmid DNA.

Float the two tubes in their foam holder in ice water for 10 minutes.

Summary
The + DNA tube contains bacteria and DNA from another source. The - DNA tube contains only bacteria.

While Waiting...
While waiting for the tubes to cool, use a marker to label your agar plates with a letter that indicates the type of bacteria that they will receive.


 * **Plate** || **Label** || **Comments** ||
 * LB/amp || T || this plate will receive transformed bacteria ||
 * LB/amp/ara || T || this plate will receive transformed bacteria ||
 * LB/amp || N || this plate will receive normal, untransformed bacteria ||
 * LB || N || this plate will receive normal, untransformed bacteria ||

Heat Shock
Heat shocking is used to make the //E. coli// cells more permeable so that they take up the modified plasmids more readily. Bring your container of ice water and the microtubes to the 42 degree water bath. Transfer the foam microtube holder containing the + and - tubes to a 42 degree C water bath for exactly 50 seconds then return the tubes to ice and water. It is important that this heat shock last for exactly 50 seconds. In order to insure that the time is exactly correct, have one person time the procedure while another transfers the tubes to the water bath and returns them when 50 seconds has elapsed.

Allow the tubes to remain in the ice water for 2 minutes. Remove the foam microtube holder containing the microtubes from the ice water and place it on your bench top. Add 250 ul of LB broth to each of the bacterial cultures (the + tube and the - tube) with a micropipette. Allow the tubes to stand at room temperature for 10 minutes.

Transfer Bacteria to the Culture Plates
Mix the two tubes by tapping them with the fingernail of your index finger. Transfer 100 ul of the solution from the + microtubule to the surface of the agar in one of the plates labeled T using a micropipette. The 100 ul line is the second one from the tip on the pipette. Transfer another 100 ul to the other plate labeled T. Use a different pipette tip to transfer 100 ul of solution from the - microtubule to the surface of the agar in one of the plates labeled N. Transfer another 100 ul to the other plate labeled N. Spread the mixture over the entire surface of the agar in one of the plates using a sterile loop. Use different sterile loops to spread the mixtures in the other plates. The diagram below shows sterile technique.

Incubate the Plates
Turn the plates over so that the agar is up and the cover is on the bottom side. Stack them one on top of the other and then tape them together. Write your name on the tape. Your instructor will transfer your plates to the 37 degree incubator in the Microbiology Laboratory. They will remain in the incubator for 24 to 48 hours.

Disposal and Clean-up
The used loops, used pipettes, foam holder, and microtubules should be disposed in a biohazard container. The agar plate containing bacteria that you used for your initial culture should also be disposed in a biohazard container. The surface of your work area should be sprayed with disinfectant and wiped down with paper towels. Don't forget to wash your hands before leaving the lab.

=Results=

Here ya go kids! Let's see what you come up with :)

AP Lab 6B: Analysis of Precut Lambda DNA Using Gel Electrophoresis
 * OBJECTIVES ** : During this lab activity, you will learn:

To use electrophoresis to separate and sort a large group of DNA molecules according to their size.

To construct a standard curve to determine the molecular weight of unknown DNA fragments when given a standard of known molecular weights for comparison.  To use data analysis software to analyze a digital image of a DNA gel produced in lab.

Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their sizes. DNA contains many negative electrical charges due to the many phosphate groups located on the outsides of the “ladder” that makes up the molecule. Scientists have used this fact to design a method that can be used to separate pieces of DNA. A solution containing a mixture of DNA fragments of variable sizes is placed into a small well formed in an **agarose** gel that has a texture similar to gelatin. This gel is then placed into a box filled with a buffer solution containing ions (for example, TAE or TBE) that aid in conducting a charge through the gel. The electric current causes the negatively charged DNA molecules to move towards the positive electrode. Imagine the gel as a strainer with tiny pores that allow small particles to move through it very quickly. The larger the size of the particles, the slower they are strained through the gel. After a period of exposure to the electrical current, the DNA fragments will sort themselves out by size. Fragments that are the same size will tend to move together through the gel and form bands. One of the basic tools of modern biotechnology is DNA splicing: cutting DNA and linking it to other DNA molecules. The basic concept behind DNA splicing is to remove a functional DNA fragment — let’s say a gene — from the chromosome of one organism and to combine it with the DNA of another organism in order to study how the gene works. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes have been given to people with nonfunctional genes, such as those who have a genetic disease like cystic fibrosis. This particular use of spliced DNA is known as **gene therapy**.
 * How Can Fragments of DNA Be Separated From One Another? **

In this laboratory activity, the DNA you will be working with is the genome from a virus that has already been cut into pieces with **restriction enzymes**. Your task will be to determine the size of the DNA pieces using a procedure known as **agarose gel electrophoresis**. This involves separating a mixture of the DNA fragments according to the size of the pieces. Once this is accomplished, you will compare your pieces of DNA with pieces of DNA whose size is already known. Of the DNA fragments that are produced, imagine that one piece in particular represents a specific gene. This gene can code for any number of traits. But before it can be given to a recipient organism, you must first identify the gene by using gel electrophoresis. You will now carry out an electrophoresis using directions given to you in class. Be sure to follow each step carefully and exactly. Here are a few notes you may want to review prior to performing the procedure: 1. The volumes of material you are working with are extremely small. Remember that you are working with **microliters** of liquid so that when you pipet, understand that you may not be able to visualize the liquids very easily, especially if they are clear. 2. The agarose gel acts as a molecular strainer with pores distributed throughout the gel, allowing for various sizes of DNA fragments to wind their way through the gel. 3. DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. A blue loading dye, composed of two blue dyes, is added to the DNA solution. The loading dyes do not stain the DNA but make it easier to load your samples into the agarose gels and monitor the progress of the DNA electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. 4. You will need to stain your gels after the procedure in order to make your DNA visible. Since DNA is naturally colorless, it is not immediately visible in the gel. Unaided visual examination of the gel after electrophoresis indicates only the positions of the loading dyes and not the positions of the DNA fragments. DNA fragments are visualized by staining the gel with a blue dye called Fast Blast DNA stain. The blue dye molecules are positively charged and have a high affinity for the DNA. These blue dye molecules strongly bind to the DNA fragments and allow DNA to become visible. These visible bands of DNA may then be traced, photographed, sketched, or retained as a permanently dried gel for analysis.
 * Procedure: **

**Results:**