BIOL2103
Biological Sciences Laboratory Course
Practical 3
Laboratory manual
Isolation of nucleic acid and spectrophotometry
Introduction:
The ability to isolate and quantify nucleic acids accurately and rapidly is a prerequisite for many of the methods used in biochemistry and molecular biology. The concentration of DNA or RNA in a sample, and its condition, are often estimated by running the sample on an agarose gel. Such concentration estimates are semiquantitative at best and are time-consuming.
For a more accurate determination of the concentration of DNA or RNA in a sample, a UV spectrophotometer is commonly used. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. The absorption maximum of DNA/RNA is approx 260nm. The purity of a solution of DNA can be determined using a comparison of the optical density values of the solution at various wavelengths. For pure DNA, the observed A260/A280 ratio will be near 1.8. Elevated ratios usually indicate the presence of RNA. The A260/A280 ratio is used to assess RNA purity. An A260/A280 ratio of 1.8-2.1 is indicative of highly purified
RNA. The 260/280 ratio below 1.8 often signal the presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is indicated by 230/260 ratios greater than 0.5.

Workflow
Time
2 days before the lab session During lab session
1:30 pm

Task
Cell culture

2:00 pm

RNA isolation

5:15 pm

Spectrophotometric analysis of your sample

Work done by
Technician

Briefing
Student

Cell culture (Prepared by technicians)

1 x 105 TM4 cells were seeded in 35mm sterile culture dish with DMEM/10%FBS medium two days before this practical session.

Isolation of RNA from cultured cells
WARNING: TRIZOL contains phenol and causes burns if swallowed and in contact with skin. Be careful!
CAUTION: When working with TRIZOL reagent, use gloves and wear lab coat.
Avoid contact with skin or clothing and avoid breathing vapor.
If accidently contact with skin, wash immediately with plenty of water and report to the demonstrator.

(i) Homogenization
1. Each student should collect a dish of cultured cells and isolate RNA from the sample individually. 2. Aspirate the culture medium and wash cells with 1XPBS twice (1ml/dish/wash).
3. Lyse cells directly in the culture dish by adding 1ml of TRIZOL reagent and pass the cell lysate several times through pipetting.

(ii) Phase separation
1. Transfer the homogenized sample into a 1.5ml tube.
2. Incubate the homogenized sample at room temperature for 5min to allow the complete dissociation of nucleoprotein complexes.
3. Add 200µl of chloroform into the homogenized sample and cap sample tube securely. 4. Shake the tube vigorously using a vortex mixer (~10-15sec) and incubate the sample at room temperature for 3min and centrifuge at 12000g for 15min.
5. Following centrifugation, the mixture separates into a lower red, phenol-chloroform
(organic) phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.

(iii) RNA precipitation
1. Transfer 400µl of the aqueous phase to a fresh tube using p200 pipettor.
Remember: Not to interrupt the interphase and organic phase to avoid DNA and protein contamination
2. Precipitate the RNA from the aqueous phase by mixing 0.5ml of isopropanol. Vortex.
3. Incubate the samples at room temperature for 10min and centrifuge at 12000g for
10min.

(iv) RNA wash
1. Remove the supernatant. Wash the RNA pellet (white pellet) with 1ml of 75% ethanol. Mix the sample by tapping.
2. Centrifuge at 7500g for 5min at 4oC

(v) Redissolving the RNA
1. Aspirate all the ethanol and air-dry the RNA pellet until the pellet turns colorless.
2. Dissolve the RNA in 50µl pre-warmed RNase-free water and incubate at 60oC for
3min. (Volume of RNase-free water is dependent on the size of the RNA pellet)
3. Pipette up and down the RNA sample several times to ensure the pellet dissolves completely. 4. Keep the dissolved RNA sample on ice and the sample is now ready for spectrophotometric analysis.

Spectrophotometric analysis of your sample
Determine the absorbance of your sample
1. Transfer 40µl of RNA sample into 1,160µl of ddH2O in a disposable UV grade cuvette, mix well and keep on ice
2. Prepare 2 blanks by adding 1.2ml of ddH2O in a disposable cuvette (for setting zero).
3. Follow the demonstrator’s instruction and determine the absorbance of your samples at 260nm and 280nm.
4. Record the absorbance and calculate the concentration of your sample and calculate the total amount of RNA isolated from your preparation. Record all the above in your report. Table 1. Accepted spectrophotometric nucleic acid conversion values
Absorbance at 260nm (=1)
Concentration
Double-stranded DNA
50µg/ml
Single-stranded DNA
37µg/ml
RNA
40µg/ml

Report
Answer all questions on the worksheet provided. Remember to show detailed calculation steps on the worksheet. Also, the original data printout (your RNA sample) from the spectrometer should be attached.