The correct answer is polymerase cycling assembly. In this reaction, multiple template sequences are included into a reaction with primers to the 5' most and 3' most sequence of the desired final product. The template sequences must also have regions of homology overlap with each other such that upon denaturing and annealing they hybridize to form a larger fragment. Upon hybridization, the primers will promote polymerase amplification of one large product.

The correct answer is the start codon should be 5' to the biochemical tag and the stop codon should be 3' to the protein of interest. When designing fusion proteins with N-terminal biochemical tags, it is important to remove the native start codon for the protein of interest to prevent initiation of transcription at multiple sites. A start codon is required immediately 5' to the biochemical tag to include the tag in the expression of the fusion protein. Only one stop codon is required at the end of the fusion protein sequence (3').

Directional cloning is the process by which two restriction enzymes cut the plasmid and the subject DNA, creating a situation in which the plasmid cannot recircularize because the only viable combination of linking is plasmid-subject DNA-plasmid.

The correct answer is the ampicillin the nutrient agar plates expired. The plasmid expresses beta-lactamase, an enzyme that degrades ampicillin. Successfully transformed bacteria will be able to grow in the presence of ampicillin, but untransformed bacteria should not grow. Since the bacteria in our control transformation do not have ampicillin resistance conferred by beta-lactamase, we would expect that no growth would be observed on any nutrient agar plates with ampicillin. However, we observe uncontrolled growth on these plates, indicating that the ampicillin has expired and is no longer a viable selectable marker for transformed bacteria.

The correct answer is introduce membrane pores. Escherichia coli grows optimally at 37 degrees Celcius. However, an increase in temperature causes the plasma membrane to become more fluid, introducing small pores through which plasmid DNA can enter into the cell.

Cloning with the Lambda phage involves all of these basic steps. The subject DNA is annealed to sticky ends inside the phage DNA. A plate of bacteria is infected with the phage, which actually does the replication of your DNA. The phages are specifically engineered to only insert DNA into the bacteria for replication if it actually incorporated your DNA fragment.

The annealing temperature is the optimal temperature at which the primers bind the template DNA sequence. This temperature takes into account several factors: the number of basepairs in the primers, the relative guanine-cytosine content, and their melting point.

The correct answer is calcium chloride surrounds the bacterial membrane and attracts negatively charged DNA. Plasmid DNA is introduced to calcium chloride incubated bacteria and are mixed at for up to one hour. Then, a heat shock to causes the plasma membrane to loosen, allowing the plasmid DNA to enter the cells. A recovery period in nutrient broth at promotes plasma membrane recovery and initiation of bacterial replication.

None of the answer choices are correct. In order to successfully clone a bacterial gene into a mammalian expression vector, the following must be completed: First, the bacterial gene should be PCR amplified from bacterial cDNA, which contains only exons. Second, the vector backbone should be digested with restriction enzymes to linearize the vector so that the bacterial gene can be inserted at a specifc locus (multiple cloning site). Third, the amplified bacterial gene and the digested vector backbone are ligated together to create the desired expression construct.

These were all important advances in improvement of the Sanger technique that increased DNA sequence output. Modification of polymerases allowed quicker thermal cycling reactions and incorporation of manufactured bases with fluorescent tags. Four different fluorescent tags for each base allowed for easy identification of different bases. Lastly, capillary chromatography allowed for huge parallelization of DNA sequencing.