Question

Question To investigate types of RNA expression in the cells, your team plans to amplify and clone nucleotides in a bacterial vector, and then check sequencing. Describe the detailed procedures to obtain nucleotide sequence of RNA expressed in cells using all of the following key words (1) Reverse transcriptase, (2) cloning, (3) PCR, (4) transformation, (5) Sanger method.

Answer 1

1) mRNA isolated from cells using oligo dT affinity chromatography.

2) Isolated mRNA will be converted into cDNA using reverse transcriptase enzyme

3) This cDNA will be used as a template for amplification of specific gene of interest which we want to clone, using gene specific primers

4) amplified gene of interest will be digested with restricted enzyme which cloned into vector

5) Now cloned vector transformed into E.coli which grown in medium containing specific antibiotic.

6) Grown E.coli used for screening purpose for having positive clone.

7) Once we got colonies having specific clone with gene of interest is confirmed using gene sequencing method which follows Sanger method.

Hope it's clear...thanks