Laboratory 2: Ligation of Dna Fragments

LABORATORY 2: 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 -galactosidase. X-gal (5-bromo-4-chloro- 3-indolyl--D-galactopyranoside), a lactose analogue, is broken down by - galactosidase to produce a product that is deep blue. Thus, if ligation is successful, lacZ’ is interrupted, a competent -galactosidase is not formed, and the colony is white. If ligation fails, lacZ’ is translated, a competent - 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--galactopyranoside) and IPTG (isopropyl- thiogalactoside), an inducer of the enzyme, for blue/white selection.

 Make 2 plates: 40 l of your transformation solution onto each plate 80 l 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.