Biochemistry of life

 

FC027 Biochemistry of life
Practical course report 2
Plant DNA extraction, PCR and gel electrophoresis
Name:
Student ID:
Tutor:——————–Introduction
“Introduce” all the techniques used – plant DNA extraction (all genetic research starts with DNA – the hereditary molecule, describe the principle of the experiment – it can be done in a very simple, basic way, using simple chemicals, hence the “kitchen sink” method or “quick and dirty”), PCR – principle, what is it used for, gel electrophoresis (what’s the principle behind the process and applications)Aims of the experiments (to obtain plant DNA, use it as template to amplify “housekeeping genes” and verify the results through gel electrophoresis.
Methods
session 31. Add the Extraction Buffer to the zip-lock bag and mash the plant material using your fist
2. Add the plant material to the zip-lock bag and mash it in the Extraction Buffer using your fist
3. Transfer 500 μl of the material into a clean 1.5ml epi tube 4. Centrifuge for 5 min at max. speed
5. Transfer 150-200μl of the supernatant to a clean microcentrifuge tube (ask your supervisor)
6. Add 300μl of cold 96% EtOH to the supernatant 7. Mix8. Centrifuge for 10 min at max. speed9. Discard the supernatant and let dry for 20 min 10. Add 50μl of TE Buffer
11. Mix12. Centrifuge for 5 min at max. speed13. Transfer 40μl of the supernatant into a clean, clearly labeled microcentrifuge tube 14. Store the DNA at -20oC (+4oC short term)
Extraction Buffer (100 ml)
page3image10384Stock
page3image11608Use
Final conc.
page3image142161M Tris Cl (pH=7.5)
page3image1556020 ml
200 mM Tris Cl (pH=7.5)
page3image180645M NaCl
page3image193285 ml
250 mM NaCl
page3image217760.5M EDTA (pH=8.0)
page3image23104 page3image235765 ml
page3image24544 25 mM EDTA (pH=8.0)
10% SDS
5 ml
0.50% SDS
page3image29176dH2O
page3image30376 page3image3126465 ml
page3image31912 TE Buffer
Total: 100 ml
Total: 100 ml
Stock
Use
Final conc.
1M Tris Cl (pH=7.5)
1 ml
10 mM Tris Cl (pH=7.5)
0.5M EDTA (pH=8.0)
0.2 ml
1 mM EDTA (pH=8.0)
ddH2O
98.8 ml

Session 41. Note the two PCR microtubes containing the reaction mix. Add 5 l of DNA to one of the samples.
2. Add 5 l of sterile dH2O to the other reaction tube. Make sure to label the tubes appropriately.
Note: The reaction mix has been prepared to the volume of 45 l, by adding 5l of DNA or H2O the final volume of the reaction will be 50 l.
The reaction mix has been prepared by the technician beforehand and it contains:
10 x PCR Buffer (– Mg) 50 mM MgCl210 mM dNTP mix10 M forward primer 10 M reverse primer Template DNA
page4image7616(you are adding this) _______________________________________________________________________________
Taq DNA polymerase Sterile dH2O
35.3 l 50 l
Final reaction volume
PCR conditions: 1. 94 ̊C2. 94 ̊C3. 56 ̊C
4. 72 ̊C 5. 72 ̊C 6. 4 ̊C
5 min
30 sec 1 min 30 sec 5 min 
Initial denaturation step
Steps 2 – 4 for 25 cycles
Final extension Permanent hold
5 l 1.5 l 1 l 1 l 1 l 5 l 0.2 l
Session 5
Arrange all your samples to be loaded on the gel in the order shown on the scheme below
2. Add loading dye to all samples (ask your supervisor about quantity).
Note: Make sure to wear nitrile gloves (blue) when handling gels!
3. When all your samples are ready, carefully load them in order from left to right.
4. Close the electrophoresis tank (pay attention to the orientation of the electrodes) and switch the power supply on.
5. Due to the time necessary to complete the process you will receive the result in your next lesson.

Describe the procedures performed in your own words.
Results and Discussion
“Discuss” the procedures and your results (use any illustrations you think might be helpful from the PDF protocols of the course and add your gel picture to comment on). For example you can mention you also isolated plasmid DNA from bacterial cells, DNA isolation follows the same principle, but this was “quick and dirty” and mini prep was done using a commercial kit, which is very “clean”. Use the “tasks” section of the protocols as a guideline of what to mention, comment and interpret your results.
Conclusion
Where your experiments successful and did you achieve your aim (mentioned in the introduction
Task – Session 3
1. How did you break down plant tissue and cells to access the DNA?2. Why is ethanol used in the procedure? 3. Why is DNA stored in TE buffer?4. Name a potential use for the DNA you extracted and explain the basic principle of the experiment.

Tasks – Session 4
1. Which genes are you amplifying by PCR and why?2. Explain the purpose of the different components of the PCR. Why are you adding H2O instead of DNA template to one of the reactions?

 

Tasks – Session 5 (both R1 & R2)
1. What is the function of the electrophoresis buffer?2. Why did you mix your samples with loading dye?3. How is DNA visualised in the gel?4. Interpret the DNA signals (bands) in your samples.