Sample exam in Molecular Biology

1. You have just isolated 0.03ng of DNA from a single human hair. You now want to amplify a single-copy nuclear gene. Do you have enough DNA for this? Explain your answer.
   

 

1. You are doing PCR using Drosophila template. If you wanted 1000 template molecules, how much DNA will you need? Note: The Drosophila genome is about 150Mb and your product is derived from a single copy sequence.
   

 

1. You want to make up 200ul of a PCR master mix that contains the following:
   

10U amplitaq gold    stock=5U/ul

1X buffer        stock=10X

2.0 mM Mg        stock=25mM

200uM dNTPs        stock=10mM of each dNTP

500uM each primer    stock=0.1nMoles/ul (20 mer)

10ng template        stock=5ug/50uls

Water to 200ul

 

How much of each ingredient do you add?

 

1. To save money, you want to order the least amount of primer possible. The company says the smallest amount you can order is 1 nM. How much master mix could you make from this? Note: Assume that your mix will contain 200uM primer and that the primer is 20 bases long (a 20 mer).
   

 

1. You want to make up 10 mls of a stock solution of 1M Tris (MW=121) and 0.1M EDTA (MW=360). The Tris is a solid and the EDTA comes from a 0.5M stock. How much of each ingredient would you need?
   

 

1. a. What is 'in situ' PCR?
   
   1. Why would you want to do it?
      
   2. How do you keep the in situ PCR product from floating away?
      

 

1. Explain what would happen to your sequencing traces if you did the following:
   
   1. Forgot to add the Exosap in the PCR product clean-up
      
   2. Forgot to add the EtOH to the sequencing reaction clean-up
      
   3. Forgot to denature the sample before loading on the gel.
      

 

1. What would happen to your PCR product (as seen on an agarose gel) if you did the following:
   
2. Misprogramed the thermocycler to do 60 rather than 30 cycles
   
3. Forgot to include the 10 min initial 94C denaturing step when using amplitaq gold
   
4. Added only one primer to the master mix rather than two.
   

 

1. What would happen to your DNA prep if you did the following:
   
2. Forgot to add the EtOH to the wash buffer
   
3. Centrifuged at full speed rather than 1/2 speed during the wash steps
   
4. Used 10mM Tris/1mM EDTA rather than water for the elution step
   

   
    

5. Your RT-PCR reaction didn't work! That is, when you run a gel you see no band of the expected size. You do see a smear below the 50bp marker and you see a very faint band 500 bp larger than your expected RT-PCR product. Outline the troubleshooting steps you would take to get the RT-PCR reaction to work.
   

   
    

6. a. Most thermocyclers have heated lids. Why?
   
   1. Would you ever not want a thermocycler with a heated lid? Explain your answer
      

 

1. Explain how dUTP and UNG can help prevent contamination in a PCR reaction. What types of contamination is not prevented by this procedure?
   

 

1. Explain the process of cycle sequencing with dRhodamine labeled dye terminators
   

 

1. When is it a good idea to sequence bulk PCR product and when is it a good idea to sequence cloned PCR products?
   

 

15. Describe two different types of probes used for real time PCR.