All of these answers are correct. Next generation sequencing substantially increased DNA sequence generation speed, and makes it much easier to assemble full genomes. Earlier, Sanger sequencing requires that you know a primer sequence for each fragment you'd like to sequence, but its read lengths are still longer than next generation sequencing methods, which rely on creation of many relatively short sequences.

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.

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 correct answer is second next-generation sequencing requires amplification of DNA, but third next-generation sequencing can sequence single molecules. This major advance in sequencing technology allows a researcher to only sequence the DNA present in a given cell, for example, with third generation sequencing. This reduces amplicon bias that can occur when amplifying DNA to prepare it for second generation sequencing, generating truly representative sequencing.

The correct answer is TAS amplifies conserved yet divergent genes, such as rRNA genes, to identify species. Many species do not have sequenced genomes. Metagenomic DNA isolation is performed regularly to survey microbial species in a given sample and can be sequenced by next-generation sequencing. However, identifying species based on whole genome sequencing is very difficult because we have not sequenced the genomes for most organisms. Furthermore, computationally aligning whole genomes to reference genomes is very tedious and time consuming. rRNA genes have highly conserved regions that allow researchers to design primers to anneal in almost every species. However, the highly divergent portions of rRNA genes allow for specie identification. TAS amplifies and sequences specific genes, such as rRNA genes, for specie identification studies.  

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'). 

The correct answer is that there is no control over the orientation of the insertion into the plasmid backbone. Since there are no single stranded base pair overhangs that normally occur with restriction enzyme digestion in a blunt cutter digestion, the insertion (with blunt ends) will ligate with the plasmid backbone (with blunt ends) in both orientations randomly. This creates a grave difficulty in analyzing clones to make sure the gene insertion is in the correct orientation. 

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.  

The correct answer is that the transformed bacteria need time to transcribe and translate the antibiotic resistant gene on the plasmid. Bacteria used in transformations do not inherently contain antibiotic resistance genes. The genes that confer antibiotic resistance for bacteria often come from exogenous plasmids introduced by techniques such as transformations. There is a lag in expression of these plasmids, and as such, the transformed bacteria will not be immediately antibiotic resistant following introduction of the plasmid.