up a PCR reaction and decide that you want to run your DNA for a certain you are
ID: 261122 • Letter: U
Question
up a PCR reaction and decide that you want to run your DNA for a certain you are sbouries for 2 hours. You program the thermocycler to run the denaturation step for 45 nt e nnealing step for 2 minutes, and the extension step for 1 minute and 15 seconds. sesearted off with 5 copies of DNA in my sample, how much DNA would I have after I run my of cycles seconds, the a ir i reaction for 2 hours? (show all calculations) Ived in the cloning of a particular gene( include components necessary for each step) 37. What are two screening methods we can use to determine if our transformed cells contain our gene of interest 38. Describe the components in a vector if transformed cells are blue in the blue-white screening process 39. Describe the components in a vector if transformed cells are white in the blue-white screening process 40. Describe Gene silencing and how the RNAi pathway is initiatedExplanation / Answer
35. question part not visible
36. Gene cloning entails detachment of specific gene or DNA fragments from a donor cell and then joining it to a short carrier molecule named vector and then replicating this recombinant vector into a host cell.
Main Steps Involved in Gene Cloning
Isolation of donor gene or DNA
Election of a suitable vector
Inclusion of donor DNA fragment into the vector
Transformation of recombinant vector into a proper host cell
Isolation of recombinant host cell.
37. 5 ways of screening:
1. Blue-white screening
Blue-white screening is an extensively used method to check successful cloning. the insert is cloned into a vector comprising a lac?? sequence which encodes the ?-peptide, a working subunit of the ?-galactosidase enzyme. The multiple cloning sites prevail inside the lac?? sequence. The plasmid must be transformed into a specific strain of E. coli which contains the lac?? ?15 mutation. An empty vector will produce blue colonies. If the vector contains the DNA insert the colonies will be white. It is likely to get false positives so additional evidence of the insert in white colonies is suggested.
2. Positive selection vector
A method to clarify screening is to utilise an accurate selection vector. Positive selection vectors provisionally reveal a lethal gene like restriction enzyme that digests the genomic DNA of the bacterial host. This expression is disturbed by ligation of a DNA insert into the cloning site. therefore, the growth is seen only in these cells with recombinant plasmids.
3. restriction digest
First, separate plasmid DNA from an overnight bacterial culture.
then, Take restriction enzymes that easily decide if the plasmid comprises an insert. digest the purified plasmid DNA from recombinant clones employing restriction enzymes. Run the digested plasmid on an agarose gel to confirm that the vector backbone and insert are of the expected sizes.
4. Colony PCR
The primers can be insert-specific, vector-specific, or both to find the insert. To learn the orientation of the insert, a mixture of vector-specific and insert-specific primers is supported. Individual colonies can be subjected to the PCR. The unused part of the colony may be used to inoculate a culture media with appropriate antibiotic for downstream applications.
5. Sequencing
38. An empty vector will generate blue colonies because the action of the ?-peptide (?-galactosidase) continues. The colourless X-gal (lactose analogue) presented in the screening plates is hydrolyzed by ?-galactosidase to produce a blue pigment (5,5'-dibromo-4,4'-dichloro-indigo).
39. If the DNA is included in the vector, the insert will disturb the lac?? sequence, then the ?-peptide will not be expressed. X-gal will not get hydrolyzed. Consequently, the colonies will be white if the DNA insert is present.
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