Tony Guo 10301016034
Lab: Extraction of Genomic DNA from Whole Blood, PCR, Gel Electrophoresis, and DNA
Sequence Analysis
Purpose: To practice and understand DNA extraction techniques, polymerase chain reaction, gel
electrophoresis, and DNA sequence analysis, along with re-confirming the point mutation of
Parkinson Disease through these techniques.
Theory: This laboratory involves various theories combined together. The DNA extraction kit
absorbs DNA in high salt concentration as well as elutes DNA under low salt concentration. We
use the kit to absorb DNA from a blood sample and remove the blood plasma and other
contents. Then, by subjecting the kit to low salt concentration, the DNA is released, allowing us
to obtain the DNA. Next, the DNA is amplified through PCR via a heat resistant Taq polymerase
to speed up the DNA replication at high temperature as well as specific primers to amplify the
section wanted. Once a sufficient amount of DNA is obtained, Gel electrophoresis can be used
to determine whether the PCR is successful or whether the sequence with known base-length
shows up on the gel. After which, the DNA sample will be sent for sequencing, and the results
will confirm whether Parkinson Disease is a point mutation.
Materials:
Genomic DNA extraction kit
Purification Resin
GN Binding Buffer
Washing Buffer
Purification Column
Ethanol
DNA Template
Primers
Taq PCR Mastermix
Taq Polymerase
Deoxynucleoside Triphosphate
Divalent Cations
ddH2O (Double-distilled Water)
Ethidium Bromide
Agarose
TAE
Buffer (TAE)
DNA sample
Method:
1. Add 0.4ml whole blood to 1ml purification resin in a tube. Invert tube 5-6 times gently and
leave to incubate for 3 minutes at room temperature. Invert the tube again at the half of the 3
minutes. Spin at 5000 rpm for 3 seconds. Discard the supernatant.
2. Re-suspend the pellet in 1 ml of GN binding buffer. Invert the tube. Spin at 5000 rpm for 3
seconds. Discard the supernatant.
3. Re-suspend the pellet in 0.5 ml of washing buffer. Invert the tube . Spin at 5000 rpm for 3
seconds. Discard the supernatant. Repeat this step again.
4. Add 0.8 ml of ethanol to the pellet and re-suspend the pellet. Transfer it to a new Purification
Column. Spin at 12000 rpm for 1 minute. Discard ethanol in the lower collection tube. Spin again,
discard ethanol completely.
5. Put the Purification Column in a new 1.5ml tube, add 100μl ddH2O to the Resin of the
Purification Column. Incubate for 3 minutes at room temperature. Spin at 12000 rpm for 2
minutes.
6. Finally, the genomic DNA is in the tube.
7. Mix template DNA with Primer 1 and 2 and ddH2O. Add Taq PCR Mastermix last.
8. Set-up the PCR machine
a. Initialization Step: 94C 8minutes
b. 35 cycles:
i. Denaturation Step: 94C 30seconds
ii. Annealing Step: 55C 30seconds
iii. Extension Step: 72C 50seconds
c. Final Elongation: 72C 10minutes
d. Final Hold: 4C
9. Dissolve 0.2g Agarose in 20ml TAE.
10. Bring the solution to boil to dissolve the agarose solid.
11. Cool the solution to 60C with stirring/swirling.
12. Add Ethidium Bromide stock into the gel solution for final concentration of 0.5ug/ml.
13. Stir the solution to disperse the ethidium bromide and pour it into the gel rack.
14. Insert the comb into one of the sides of the gel about 5-10mm from the end of the gel.
15. When the gel has cooled down, remove the comb. The holes are the wells or slots for the
DNA.
16. Put the gel, together with the rack, into a tank with TAE. The gel must be submerged in TAE
and the wells/slots must be at the negative terminal.
17. After the gel has been prepared, use a micropipette to inject about 3uL of stained DNA.
Close the lid of the electrophoresis chamber and apply current.(100V for 30 minutes). The
coloured dye in the DNA ladder and DNA samples acts as the “front wave”. The current should
be stopped when the front wave reach the end of the gel.
18. The DNA is stained with ethidium bromide and thus visible under ultraviolet light.
19. Once the PCR is confirmed to be successful, we can send the sample for DNA sequencing by
a bio-technology company.
Results and Analyzation:
The results of the electrophoresis shows a dark band at the same level with the control with the
same base length readings from both the ladder and the control. This is because the primers
have successfully amplified the target genes, thus making the PCR a success. If the PCR fails,
then we would be observing bandless gels or multiple-banded gels. Therefore, we sent the
target sequence to a bio-technology company for DNA sequencing, and from the sequencing
results, we can confirm that Parkinson Disease patient has a point mutation at Parkin (Park2)
Exon4, which is a mutation from AGC to AAC.
Sources or error:
1. Contamination of the DNA.
2. Stopping the gel electrophoresis at the wrong time.
3. Incorrect removing of the comb, creating crooked wells, for the gel results.