Experiment:+UV.Spectrophotometry+of+DNA+

Aim+Using UV-spectrophotometry, determine the identity of a nucleotide, concentration of DNA and protein contamination and investigate the hyperchromic effect of dsDNA.

Experiment+1:+

Materials)2 quartz cuvettes 3 mL distilled water 3 mL unknown solution Pipette Spectrophotometer

Method)1. Add 3 mL distilled water to one cuvette and 3 mL unknown solution

containing the base in the other. 2. Baseline correction: H2O on spectrum mode

Absorption: in photometric mode measure abs from 230-310 nm. Blank each time wavelength is changed.

Reference: MBLG2071 Lab Manual, pg. 21.

Results)Solution D was thymine.

+ +

!0.2%

!0.1%

0%

0.1%

0.2%

0.3%

0.4%

220% 240% 260% 280% 300% 320%

Absorbance*

Wavelength*(nm)*

Absorption*Spectrum:*Solution*D*

Experiment+2:+

Materials)3 test tubes 2 quartz cuvettes Tris-EDTA buffer (TE buffer) DNA solution Spectrophotometer

Method)1. Calculate amount of unknown DNA solution must be added to 3 mL

quartz cuvette containing TE buffer. Consider the concentration range and make up 3 replicates.

2. Baseline correction: TE buffer on spectrum mode. Absorption: in photometric mode measure abs at 260 nm and 280 nm.

Reference: MBLG Lab Manual, pg. 22-23

+Experiment+3:+

Materials)3 test tubes 2 quartz cuvettes Tris-EDTA buffer (TE buffer) Phosphate formaldehyde buffer DNA solution 80ºC water bath Ice bath Spectrophotometer

Method)1. Calculate dilution needed to make the absorbance at 260 nm between

0.4 and 0.6. 2. Dilute DNA with phosphate formaldehyde buffer. 3. Make up a blank with the same proportions of phosphate formaldehyde

buffer and TE buffer as the diluted DNA. 4. Dispense 3 mL aliquots of the diluted DNA into two test tubes and

cover with cling wrap. 5. Place one test tube in the 80ºC water bath and at room temperature for

10 minutes. 6. Once incubated for 10 minutes, remove and place into ice bath for 5

minutes. 7. Remove from ice bath and let samples equilibrate to room temperature. 8. Measure absorption at 260 nm, blanking with TE buffer and phosphate

formaldehyde mixture. Reference: MBLG Lab Manual, pg. 23