You wish to insert a gene in the plasmid below. (Note the neon green lines represent restriction sites.)
Diagram
Description automatically generated
How would you create a cDNA library for the chromosomal DNA containing the gene of interest?
To create a cDNA library for the chromosomal DNA containing the gene of interest, the following steps can be followed:
1. Isolate the chromosomal DNA from the organism or tissue of interest.
2. Extract the RNA from the same organism or tissue, using a method that preserves the integrity of the RNA.
3. Convert the RNA to cDNA using reverse transcription, which uses the enzyme reverse transcriptase to synthesize complementary DNA (cDNA) from RNA.
4. Fragment the cDNA to insert into the plasmid. This can be done using restriction enzymes that cut at specific sequences, or by randomly fragmenting the cDNA and using linker sequences to attach it to the plasmid.
5. Ligate the cDNA fragments into the plasmid using DNA ligase, which joins complementary ends of DNA fragments together.
6. Transform the plasmid into a suitable host organism, such as bacteria, using a method such as electroporation or heat shock.
7. Select for transformants that contain the desired cDNA library by using a selectable marker on the plasmid, such as antibiotic resistance.
8. Screen the library for the gene of interest using a method such as a hybridization probe or PCR amplification with primers specific to the gene.
9. Amplify the library to obtain large quantities of the gene for further study or use.