Quantitative Analysis of RNA
Aim:
To determine the amount and concentration of RNA sample isolated from bacterial cells
Ligation of DNA fragments
Principle:
This experiment is purely an application of the Beer Lamberts’ Law which states that the concentration of the sample is directly proportional to the absorbance of light done by the sample. It is given by the following expression
A = E*C*l
The device UV spectrophotometer works on this principle and used to find the concentration of the sample
Materials Required :
- RNA sample
- TE buffer
- UV spectrophotometer
PROCEDURE:
Remove a 10 µl aliquot of total RNA and dilute with 990 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.0).
Restriction Enzyme Digestion
Read at A260 and A280 blanked against TE buffer and calculate the amount of RNA obtained. The RNA obtained may be determined by the formula
Total RNA (ug) = (A 260) (40 ug) (100) (0.05 ml)
- A260 is the absorbance of the solution at 260 nm
- 1 OD of RNA equals to 40 ugs/ml/A 260
- 100 is the dilution factor, and 0.05 ml is the total volume.
- Concentration = Total ug/50 ul = ug/µl or mg/ml
Results :
More.Exp…
