4. You want to clone a 1.5 kb cDNA. Which vectors discussed would be appropriate

Question

4. You want to clone a 1.5 kb cDNA. Which vectors discussed would be appropriate to use? Which would be

inappropriate? Explain your answer.

5. You want to make a genomic library with DNA fragments averaging about 85 kb in length. Which vectors discussed

would be appropriate to use? Which would be inappropriate? Explain your answer.

6. Describe the process of cloning a DNA fragment into the multiple cloning site of the vector pUC18. How would you

screen for clones that contain an insert? Explain the underlying principle between blue-white screening.

Explanation / Answer

The cloning vectors which are discussed (Options) are not mentioned in the question. however, I will answer the question based on the general cloning vectors and their sizes.
a. Plasmid vector - 0.1 to 10 kb insert.
b. Bacterial artificial chromosome - insert size 35 - 300 kb
c. Yeast artificial chromosome - insert size 100 - 1000kb
d. bacteriophage - 4 - 5 kb
e. cosmid - insert size 30 - 50 kb

4. The insert size is 1.5 kb. So the plasmid vector is most suitable for such cloning. Oher vectors have higher size limits which puts them at the disadvantage while cloning small fragments.

5. 85 kb. The most suitable is BAC. The BAC size range is 35 - 300 kb. This makes them ideal vector. The YAC has larger insert capacity and others have lower. Hence BC is the best choice.

6.
The cloning of the insert into MCS site of the vector Puc18. The PUc 18 MCS has sites for several restriction enzymes. '
- restrict the puc18 with two restriction enzyme, say HindIII and BamH1.
- insert is engineered to contain the restriction sites of the Hind IiI and BamH1. This is also restricted to same enzymes to create sticky ends.
- ligate the puc18 and insert.
- The ligated mixture is checked on the gel to obtain a single band. The unligated will give two different bands on the gel.
- The insert is ready inside the puc18.

Blue-white screening.
The successful cloning depends on the ability of detection. The recombinant colonies must be differentiated from the wild-type ones in order to determine the efficiency of cloning and to carry out further characterization. The most commonly used technique for detection of recombinant colonies is Blue/White screening.
The host contains the half coding region of lac z gene which encodes or b-galactosidase. The remaining half region of the lacZ gene is present on the puc18 vector. Upon recombination, the lacZ region from both plasmid and host can complement each other and produce the normal lacZ gene product, which will digest the lactose. The lac
In this system, an inducer of lac operon IPTG is added. Instead of lactose, a non-digestible analogue of lactose, X-gal is added in the media. In the presence of IPTG, the host synthesizes both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into the MCS located within the lacZ gene inactivates the enzyme and the enzymes do not show alpha-complementation. Bacteria carrying recombinant plasmids, therefore, give rise to white colonies

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