Brebeuf Jesuit Biotechnology

Pre-Lab Question:

1. Consider the Plant Enzyme lab.  How did we quantify an unknown amount of amylase in a barley seed?  How do you think we will quantify an unknown amount of DNA in a calf liver cell?

Part A: Homogenization

1. What is meant by the term “homogenization”? 
2. Why was the calf liver frozen prior to placing it in the blender?                                                                                                 
3. Why was the calf liver placed in the blender?                                                                                                                                                                                                 

Part B: Extraction

1. Why did you add cold 10% TCA to the liver solution?                                                                                                                                                                                                                                                                                                      TCA is a strong acid.  Watch this movie for some insight: http://jchemed.chem.wisc.edu/JCEsoft/CCA/pirelli/pages/cca5denature1.html                                                                                                                   
2. What is the purpose of centrifuging the liver solution?  Why shouldn’t you use your hand to stop the centrifuge?  Watch this short video to help answer the previous quesitons: http://jchemed.chem.wisc.edu/JCEsoft/CCA/pirelli/pages/cca6centrifuge.html                                                                                                               
3. After centrifuging the liver solution (with cold 10% TCA), did you save the pellet or supernatant?  What was in the pellet?  What was in the supernatant?                                                                                                        
4. Why did you resuspend the pellet when adding 95% ethanol to the test tube?                                                                                                         
5. Why did you add 95% ethanol to the liver solution?                                                                                                                                                             
6. After centrifuging the 95% ethanol-liver solution, did you save the pellet or supernatant?  What was in the pellet?  What was in the supernatant?
7. Why did you add room temperature 5% TCA to the liver solution?                                                                                                                                       
8. Why did you heat the 5% TCA-liver solution in a 90 degrees Celsius water bath for 15 minutes?                                                                                                                                      
9. After centrifuging the 5% TCA-liver solution, did you save the pellet or supernatant?  What was in the pellet?  What was in the supernatant?                                                                                                                                   

Part C: Extraction

1. What was the purpose of adding diphenylamine to the DNA standards (test tubes 1-4) and the unknown in test tube 5?                                                                                                                                                                                                                                                                                  
2. Why did you make DNA solutions of known concentration?  Which DNA standard (test tube 1, 2, 3, or 4) contained the least amount of DNA?  Which one contained the greatest amount of DNA?  Which standard contained the lightest shade of blue?  Which standard contained the darkest shade of blue?  Which standard contained the lowest absorbance value?  Which standard contained the highest absorbance value?                                                                                                         
3. What is the relationship between shade of blue and the amount of DNA in the sample?
4. What is the relationship between absorbance and the amount of DNA in a sample?
5. How will you determine the amount of DNA in your unknown? 

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