Loading buffer can be filter sterilized by passing through 0.22µm filter. It can be aliquot into the small volume and store at -200C.
10. Autoclave gloves
11. 550C water bath
12. Horizontal Gel electrohpresis system
13. Power supply
14. RNase free container for staining and destaining
15. Shaker
16. UV chamber
17. Gel Doc
18. Flask for preparing agarose gel
Methods:
1. Isolation of mRNA- The structure of a typical mRNA is given in Figure 18.1. it has a n-terminal CAP structure, coding sequence and a c-terminal poly A tail. The Nucleotide A forms 2 hydrogen bonding with nucleotide T and this pairing is very specific. Exploting this feature, m-RNA population can be isolated from RNA pool using a poly-T affinity column. The Steps in m-RNA isolation from cell is given in Figure 18.2. it has following steps:
1. Release of total RNA either by a lysis buffer containing detergent or by homogenization in the case of hard tissue.
Figure 18.1: Structure of a typical mRNA.
2. Mixing of poly-T containing beads with the toral RNA prepration. Due to mutual exclusive affinity, mRNA binds to the poly-T beads.
3. Wash the beads with washing buffer to remove non-specific cross contaminating species.
4. Elute the mRNA from beads and its purity can be checked on polyacryalamide gel (discussed later in future lectures).