AYou want to create and amplify a cDNA library of the genes

A)You want to create and amplify a cDNA library of the genes expressed in a certain cell type. Describe the steps and ingredients needed?

B) How would the procedure change if you wanted to only clone one single gene instead? What additional information would you need?

Solution

A) cDNA is basically created from all the mRNA\'s in the cell using reverse transcriptase.

the first step is to extract the mRNA\'s from a cell by distinguishing it from the other RNA\'s. It is generally isolated by the use of poly A tail at its end. it is eluted out by using a column purification method.

once mRNA is purified, it is coupled with a complimentary primer that binds to the poly A tail and provides a free 3\'OH end. the enzyme reverse transcriptase comes and extends a new DNA which is complimentary to the mRNA, the resulting mRNA is digested by using RNase.

this leaves us with a single strand DNA which is made into a double strand by using DNA polymerase. this DNA can then be cloned into different bacterias using restriction endonucleases and DNA ligase and then the se bacteria could be selected through screening by antibiotics.

these stocks of selected bacteria can be grown and compiled to make a cDNA library.

B) If we want to clone only a particular gene, then we will design a soecific probe just for it which has a 3\'OH end that can lead to the formation of a single stranded DNA at first and then can later be transformed into a double stranded DNA molecule.