Laboratory 2: Electrophoresis, Ligation and Bacterial Transformation
The TAs will amplify a gene of interest using PCR and supply you with the amplified DNA for ligation.
Exercise 1. Ligation of the PCR product into the TOPO vector.
For ligation, we will use the TOPO TA cloning kit by Invitrogen. It allows for efficient insertion of PCR products into a plasmid vector in a single step, at room temperature, in 5 min. It relies on the phenomenon that Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector supplied with the kit has single, overhanging 3’ deoxythymidine (T) residues that allow PCR inserts to ligate efficiently with the vector.
See attached protocol
Be sure to include a vector only control: a ligation reaction with no fragment DNA. This will give you an idea of the background ligation (the vector religating without insert). This could be prevented by dephosphorylation of the linearized vector but doesn't always work completely.
In general, for ligation of DNA fragments into a plasmid, the ligation mixture should include a 3:1 molar ratio of DNA fragment: plasmid DNA.
Ligation of DNA in a mixture routinely results in a variety of potential products.
1. Only circularized plasmids will survive in the bacteria. This eliminates all ligation products that are not recircularized.
2. In addition to our gene of interest, the vector plasmid contains an ampicillin resistance gene. We will grow the bacteria on nutrient agar containing ampicillin. This will eliminate extraneous bacteria as well as any circularized products without the appropriate antibiotic resistance.
3. Blue/white selection (Lac selection) – in addition (or instead of) dephosphorylation to eliminate religation of the vector, blue/white screening can be used. The multi cloning site in the plasmid interrupts a lacZ’ gene. lacZ’ codes for part of the enzyme b-galactosidase. X-gal (5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside), a lactose analogue, is broken down by b-galactosidase to produce a product that is deep blue. Thus, if ligation is successful, lacZ’ is interrupted, a competent b-galactosidase is not formed, and the colony is white. If ligation fails, lacZ’ is translated, a competent b-galactosidase is formed, and the colony is blue.
Once ligation is completed, transformation of E. coli will allow for amplification of the product (or products). After the ligation products have been amplified (so we have something to work with), we could do a plasmid DNA extraction and a series of restriction digests to determine if we accomplished our goal of inserting the gene of interest into the vector. These digests are routinely referred to as diagnostic digests. Unfortunately, we will not have time to do this.
Exercise 2. Bacterial transformation
Why and how bacterial cells can be made to be competent for DNA uptake is not understood. Fortunately, they can be made competent, and they can be stored in this state indefinitely at -80C.
Bacteria can be transformed in a variety of ways. Some cells are chemically competent and required only the appropriate chemical to be transformed. Others are competent for electroporation and require an electrical shock to create pores in the membrane. Still others are competent for transformation by heat shock.
We will use heat shock for transformation of our ligation product. The DNA does not need to be purified at this stage. The ligation mixture will not affect transformation by heat shock.
Depending on the cells and the source of the cells, techniques for transformation may vary slightly. Always follow the instructions on the package insert to perform your transformation.
See attached protocol
The last step of the transformation procedure is to plate the mixture onto nutrient plates with antibiotic selection (We will use ampicillin.) and incubate overnight at 37C.
· You will be provided two types of plates: 1) those with X-gal (5-bromo-4-chloro-3-indolyl-b-galactopyranoside) and IPTG (isopropyl-thiogalactoside), an inducer of the enzyme, for blue/white selection.
· Make 2 plates: 40 ml of your transformation solution onto each plate
80 ml of your transformation solution onto each plate
Colonies should appear by the next day. The TAs will put move your plates to 4oC. You should come in sometime Sunday afternoon to select colonies and make 3ml cultures to use on Monday.
Exercise 3. Gel Electrophoresis of gDNA
The standard method for separating DNA fragments is electrophoresis through agarose gels. DNA is applied to a slab of gelled agarose and then an electric current is applied across the gel. Because DNA is negatively charged (phosphate groups in the backbone), it migrates through the gel towards the positive electrode. The rate of migration depends on:
1) the size of the fragment; the smaller the DNA, the faster it "runs" through the gel;
2) the concentration of agarose; the higher the concentration, the more the agarose retards the movement of the DNA fragments; and
3) the voltage applied to the gel; the higher the voltage the quicker the DNA "runs" (but at a trade-off--the DNA fragments do not separate as efficiently).
a. Pouring the gel
A demonstration on preparing and running an agarose gel will be given. Each group should have a gel apparatus and cover, two end walls, two combs and a plastic gel tray. The gel tray should be placed in the chamber and the two end walls inserted. A comb should be placed near one of the end walls.
Make 100 ml of a 1.0% agarose in 1X TBE for your gel in a 250 ml flask. Minigels require ~30 ml agarose.
Use the microwave to melt the agarose. Be sure the solution is completely melted and homogenous.
Warning!! Never leave the agarose solution unattended when using the microwave. The solution must be frequently swirled during the heating process to prevent superheating of local areas. Always use "hot hands" or autoclave gloves when heating the agarose.
You will be provided Ethidium Bromide (EtBr) at a concentration of 1 mg/ml in a 1.5 microfuge tube. This tube should be saved and stored at 4ºC.
Warning!! Ethidium bromide is a mutagen. Remember to wear gloves when working with EtBr and to dispose of contaminated tips in specially marked containers. Gels should be placed in a plastic bag for disposal. Microwaving EtBr produces harmful volatiles.
Add EtBr to a final concentration of 0.1 to 0.5 mg/ml to agarose immediately before you pour the gel. To do this, label a 50 ml conical tube EtBr/agarose. Add the EtBr to the tube and then pour about 30 mls of the agarose into the tube. Cap and invert a few times. The agarose solution should be cooled to about 50ºC (a temperature at which one can hold the flask, but it is uncomfortable) before pouring the gel. Pouring the gel when the agarose is too hot may damage the gel apparatus.
Pour the 1.0% agarose minigel with 0.1 mg/ml EtBr and allow it to solidify.
Practice loading a few lanes with loading dye if you've never loaded a gel before. This can be run into the gel without affecting the migration of DNA added to the well at a later time.
At first it will be easier to load the wells dry. Add 1X TBE buffer to submerge the gel prior to electrophoresis. Later, you should become adept at loading wells submerged in buffer.
b. Loading the Samples and Running the Gel:
To a new eppendorf or on parafilm, add 5 ml loading dye to 15 ml of your sample and finger flick. Load 20 ml total volume of your samples per well for minigels with 8-well combs. Load 10 ml of the molecular weight markers provided. Your gel should contain:
1. molecular weight markers
2. gDNA group 1
3. gDNA group 2
4. gDNA group 3
Run the gel at 100 V until the bromophenol blue is about 3/4 through the gel (approx. 1 h).
Using gloves, carefully remove the tray with the agarose gel. Take it to the UV transilluminator and slide the gel off the tray onto the lamp. Wearing UV-protective goggles (NOT YOUR SAFETY GLASSES), examine the gel. The cover of the transilluminator may also be used to protect your eyes. Photograph your gel with a ruler for your notebook.
Conclusions.
What did your digests tell you?
Did you get the desired insertion?