Lab Manual

CLB 10003 Biology Jan 2013

SECTION OF BIO-ENGINEERING
TECHNOLOGY, UniKL MICET

EXPERIMENT 6: ELECTROPHORESIS AND DNA QUANTIFICATION

Objective: To learn the basic methods used to check the quality of extracted DNA and to
further with DNA quantification
Solutions required
1) 10 X TBE (Tris Borate EDTA). Generally diluted to 1X for use:
2) Sample DNA (from previous lab)
3) 1X TBE agarose gel (1%)
4) Lambda HindIII DNA Marker/ladder
5) Ethidium bromide (EtBr) : HAZARD!!! Wear double gloves when handling.
Experiment Procedures
A.

Electrophoresis of a DNA sample of unknown concentration with a known

standard.

1. Place the gel plate into gel mould, position the comb and ensure that the gel is
horizontal check with a spirit level if necessary.
2. Prepare a 1% agarose gel in 40 ml of 1x TBE. Heat the mixture in a microwave
oven until completely dissolved
3. Pour agarose onto gel tray and allow it to set for least 20 min.
4. Place the gel into the electrophoresis tank. Carefully remove the comb and pour 1x
TBE (same as the buffer that was used to make gel) until the gel is completely
covered.
1

Lab Manual

CLB 10003 Biology Jan 2013

5. Mix 2 l loading dye with 5 l DNA and load it into the well.
6. Mix 1l of Hindlll DNA marker with 1l of loading dye and then load it into one
of the wells.
7. Run the gel at 70 V for one hour or until the dye is about 2/3 from the origin.
8. Stain the gel with ethidium bromide (1 l ethidium bromide in 100 l ddH 2O
(HAZARD!!!!) for 10 minutes.
9. Stain the gel with distilled water for 10 minutes.
10. Illuminate the gel with UV light (CAUTION UV LIGHT IS HAZARDOUS!!!
WEAR MASK OR UV PROTECTION GLASSES IF EXPOSED TO UV
LIGHT).
11. Photograph the gel under the UV.
12. Compare the intensity of the DNA bands of the samples with the intensity of the
DNA Marker bands.
13. Quality of extracted DNA can also be assessed by looking at the gel. Good quality
DNA will show as a sharp intense band. Degraded DNA extracts will show various
degree of smearing. (Refer to figure provided below).

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Figure 1 : Agarose gel analysis of genomic DNA isolated from fish tissue.
(From left to right) Lane 1 & 2: Good DNA quality; Lane 3 & 4: DNA with
RNA; Lane 5 & 6: degraded DNA.

Lab Manual

B.

CLB 10003 Biology Jan 2013

Spectrophotometric determination of DNA concentration

Dilute 3 l of DNA with 150 with deionised water and read at A230, A260 and A280. The
A260/A280 ratio provides an estimate of the purity of the DNA. In a pure sample, this ratio
is approximately 1.8. Lower values indicate protein or phenol contamination. A230 should
be less than A260 and may be the same as A280. High A230 reading indicates that residual
phenol remains in the preparation. An A260 of 1 corresponds to approximately 50 l/ml of
double-stranded DNA in a 1 cm quartz cuvette. Nucleic acid concentration is calculated
as follows:

DNA concentration (ng/l) = A260 X 50 X dilution factor (150 l/3 l)

DNA purity = A260/ A280
Table 1: Common problems in DNA extraction and appropriate solutions.

Discussion
Calculate the DNA concentration and DNA purity.