Biology Prac Report Example

Aim:
The aim of this experiment is to investigate the process of Electrophoresis and successfully analyse DNA fragments.
Hypothesis:
That the experiment will show the visual representation of DNA proteins, and that the shorter the band the further is will travel.
Background:
Restriction enzymes are DNA-cutting enzymes found in bacteria, which cut DNA into smaller fragments. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides, known as restriction sites. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences usually vary between 4 and 8 nucleotides, and many of them are palindromic, meaning the base sequence reads the same backwards and forwards. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose gel. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins.
Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel. Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel, and identified using a DNA protein ladder.
A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. A gel pattern is used to examine DNA bands, where where the molecules in the gel can be stained to make them visible. After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation. The ethidium bromide fluoresces reddish-orange in the presence of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white. Figure 1: Gel Electrophoresis diagram Figure 2: Electrophoresis Tank and view of experiment

Equipment:
Goggles Incubator
Apron Agarose Gel
Gloves Gel tray and comb
Micropipettes Electrophoresis Tank
Minicentrifuge Power supply for tank
Vortex λ Bacteriophage DNA
Disposable tips Tip Tray Eco RI (restriction enzyme)
Water filled container for used tips 10x buffer (EcoRI)
BamHI (restriction enzyme) 10x buffer (BamHI)
HindIII (restriction enzyme) 10x buffer (R+)
Distilled water TAE Buffer solution
Staining card UV light viewing chamber
Procedure:
Day 1: DNA Digestion
1. Equipment gathered and safety procedures followed.
2. Eppendorf Tubes were labelled 1-4.
3. Correct amounts of each component were placed into each Eppendorf tube as per table 1.
4. A separate pipette tip was used for each component to avoid contamination.
5. The solution was placed on the inner side of each Eppendorf tube.
6. Eppendorf tubes were flicked, and centrifuged for 30 seconds.
7. Solutions were placed in incubator at 37°C and refrigerated for 24 hours

Day 2: Gel Electrophoresis
1. Mixtures had 2 uL loading dye added
2. 10uL of each mixture was loaded into separate wells of agarose gel
3. 20 uL of precut DNA was placed in water bath for 5 minutes at 65°C, then cooled on ice for 3 minutes.
4. 20uL of DNA ladder was loaded into separate wells
5. TAE buffer solution was added over agarose gel and halfway up tank
6. Tanks were connected to power station for 35 minutes at 100V
7. Gels photographed and stained by lab assistant
Day 3: DNA Fragment analysis
1. Photographs of gels were studied and results were recorded
2. Fragment sizes were calculated using restriction maps and gel patterns

Table 1: Measurements for experiment
Component to be added Tube 1 (control) Tube 2
(Bam HI) Tube 3
(Eco RI) Tube 4
(Hind III)
Lambda bacteriophage DNA 8uL 8uL 8uL 8uL
Eco RI - - 5uL -
10x buffer (EcoRI) 5uL - 5uL -
Bam HI - 5uL - -
10 X buffer (BamHI) - 5uL - -
HindIII - - - 5uL
10 X buffer (HindIII) 10uL 5uL 5uL 5uL Results:
Table 2: Restriction map results
Restriction Maps DNA Bam HI Eco RI Hind III MWM DNA Ladder
Number of fragments 1 6 6 8 13 10
Fragment sizes (biggest to smallest) 48502 base pairs long 21226
7421
5804
5643
4878
3530 16841
7233
6770
6527
5626
555 23130
9416
6557
4361
2322
2027
564
125 2000
5210
4988
4241
3765
2070
1990
1630
1415
9780
7645
3072
1020 10000
9000
8000
7000
6000
5000
4000
3000
2000
1000

Table 3: Gel Patterns
Gel Patterns Bam HI Eco RI Hind III MWM
Number of bands 4 6 7 11

Analysis of results:
The results obtained in tables 2 and 3 corroborated with those studied in class, and match other sources. Some of the number of base pairs in MWM for the restriction map is incorrect at times. This can be rectified by closely analysing the DNA ladder more carefully. The results from the agarose gel clearly show a visual representation of the data and were used successfully to match base pairs.
Discussion:
During this experiment errors were discovered as some gel walls were punctured which contaminated other walls. For this reason my results were taken from group 6, as they had the best results. The bands closest to the wells are the most illuminated as they are the largest bands, and therefore travel a shorter distance than that of the shorter bands. Another factor which may have affected the results is that the bands may have come up clearer had they had another hour. If this was done the results would be more accurate and easier to identify.
If this experiment was to be done again, tips would need to be changed between trials to avoid contamination. Practise at loading the wells needs to be undertaken to ensure that the wells are not broken in the future or punctured. A closer analysis of results is needed to further examine the results and to get the most accurate and detailed results.
Overall this experiment investigated the process of electrophoresis and showed a visual representation of DNA extraction and restriction enzymes.
Sources:
NOAA Ocean Exlporer. (August 26, 2010) From Ocean Explorer Webmaster, retrieved 2/5/2013. From: http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html
Gel Electrophoresis.(N.D.) From: Sdmesa.edu, retrieved 2/5/2013.From: http://classroom.sdmesa.edu/eschmid/Lab12%20-%20Biol210.htm
Gel Electrophoresis Virtual Lab.(2013). From: The University of Utah. Retrieved 2/05/2013, from: http://learn.genetics.utah.edu/content/labs/gel/