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.

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 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. 

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. 

The correct answer is cDNA. Given that this gene most likely has several introns, complementary DNA (cDNA) should be used as a template since it only contains exons. When subcloning a gene into an expression vector, only coding sequence should be present because the transcribed RNA will not be processed like it is in host cells.

Plasmids are circular pieces of DNA that are resistant to exonucleic degradation (exonucleases cut from DNA ends). Bacteria harbor native plasmids that replicate independently from their genome and express genes that often confer a survival advantage. Scientists often clone genes into plasmids and express them in a variety of host cells. 

Frederick Sanger invented the chain termination method of DNA sequencing in 1977, which has been widely used since its invention for short DNA sequences. The method is know as Sanger Sequencing. Leroy Hood was responsible for automating this method, Kary Mullis was the inventor of the polymerase chain reaction to create copies of DNA, E. M. Southern lent his name to another DNA study method called Southerns, and Craig Venter was responsible for other later DNA sequencing methods.

Polymerase chain reaction requires two, not one, synthesized primer molecules to correctly amplify a DNA molecule into new copies. One primer attaches to each end of your desired DNA segment, and amplification occurs from these sites by polymerase molecules. The other options are all correct facts about PCR.

Cosmids could handle DNA insert sizes of 25-50kb. BAC's could handle insert sizes of 100-300kb, and YAC's even more. These are still used for tricky genome projects due to this large insert size, like antifreeze protein genes in Arctic fish. Plasmids can generally only handle insert sizes of 5-10kb.

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.