Question

1. In the methods section that describes the construction of the cDNA library the authors state that they used poly(A) enriched RNA as a template to make the cDNA. What does this mean, and what would be a method that they could use to enrich/isolate poly(A) RNA? I am looking for more than just "they used the BRL mRNA isolation system". Describe how this system probably works. 2. In lab we used a plasmid as a vector, but in this article the authors decided to use a viral vector. Why do you think that they used a viral vector (agtil) rather than a plasmid? As a hint, the clone that they identified was 1859 base pairs long.

Answer 1

For cDNA construction from mRNA which has a poly A tail, we can use poly dT primers for carrying out reverse transcription reaction to produce cDNA. Poly dT primers will bind to poly A tail and only amplifies mRNAs.

To isolate poly A mRNAs we can use a column with oligo dT attached to polystyrene beads or magnetic beads. The sample can be added to this column and incubated for the poly A to bind. Later after several washing steps centrifugation can be done to collect the beads and resuspend them in elution buffer in high temperature (65°C). After another round of centrifugation , the supernatant containing Poly A RNA can be collected.

Q2

Plasmid vectors have low carrying capacity compared to that of viral vectors. Lambda gt11 has a carrying capacity of 7.2kb which means it can take up a DNA fragment that is 7.2kb in size. So here since the fragment is 1.8kb in size, viral vector is a better choice than plasmid vectors.