Lab Report Genetic Experiment 3&4

Lab Report Genetic Experiment 3&4

Name: Momoe Nasuno

Student ID: 10301016066

Major: M.B.B.S.

Group: 4

Date: 2011/12/15

Objective:

The objective of these two experiments is to first extract genomic DNA from whole blood of a Parkinson patient, amplify copies of the special Parkinson disease gene through PCR, then analysis the DNA after agarose gel electrophoresis under ultraviolet lamp.

Procedure:

Extract genomic DNA from whole blood

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 supernatant.

2. Re-suspend pellet in 1 ml of GN binding buffer, Invert the tube. Spin at 5000 rpm for 3 seconds. Discard supernatant.

3. Re-suspend pellet in 0.5 ml of washing buffer. Invert the tube. Spin at 5000 rpm for 3 seconds. Discard supernatant. Repeat this step again. (The pellet looks cream in color.)

4. Add 0.8 ml of ethanol to pellet and re-suspend pellet. Transfer it to a new Purification Column. Spin at 12000 rpm for 1 min. 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, and incubate for 3 minutes at room temperature, Spin at 12000 rpm for 2 min.

6. Finally, the genomic DNA is in the tube.

PCR

7. Add material needed for PCR to the tube contains DNA:

126ul of ddH2O

150ul of 2XTaq PCR MasterMix

6ul Primer 1

6ul Primer 2

Mix them well and divide the solution into six groups.

8. Put them into the PCR thermal cycler and operate.

Initialization step: 94℃, 8min

35 Cycles of: Denaturation step 94℃, 30s

Annealing step 55℃, 30s

Extension step 72℃, 50s

Final elongation: 72℃, 10min

Final hold: 4 ℃

Agarose gel electrophoresis

9. Make a 1% agarose solution in 20ml TAE. （0.2g agarose in 20ml TAE ）

10. Carefully bring the solution just to the boil to dissolve the agarose.

11. Let the solution cool down to about 60 °C at room temperature. Stir or swirl the solution while cooling.

12. Add ethidium bromide stock in the gel solution for a final concentration of 0.5ug/ml. Be very careful when handling the concentrated stock.

13. Stir the solution to disperse the ethidium bromide, and then pour it into the gel rack.

14. Insert the comb at one side of the gel, about 5-10mm from the end of the gel.

15. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots.

16. Put the gel, together with the rack, into a tank with TAE. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current.

17. After the gel has been prepared, use a micropipette to inject about 3µl of stained DNA (a DNA ladder is also highly recommended).

18. Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes). The colored dye in the DNA ladder (molecular weight markers) and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped.

19. The DNA is now visible under ultraviolet light.

Result:

Sequence results:

The Parkin exon4 sequence of the patient Normal control

Compare the results of the sequence of the patient and the control; we can see there is a single point mutation, where it changes the base G to A, cause the amino acid cordon change from AGC to AAC.

Agarose gel electrophoresis:

Since the DNA is stained with ethidium bromide, we can clearly see it under ultraviolet light. In this picture, the left most lane is a DNA-ladder containing DNA-fragments of 100, 200, 300, 400,500 and 600 bp. From this, we can tell the gel band of the rest six lanes indicated the DNA of the rest six groups all have the length of about 300 base pairs. Thus, we have got the correct base pairs.

Discussion:

From these two experiments, we’ve learned about how to extract DNA from the whole blood, make numerous copies of the specific gene using PCR machine, and then run the agarose gel electrophoresis and how to analyze it. This would be really useful for our future career, since we can extract the DNA from a potential patient’s blood, run the electrophoresis. By comparing the DNA with a healthy human DNA sample, we can confirm the presence of a disease in the patient.