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BIOT251 Review sheet Exam 2
Exam date: Friday April 11th, 2008 12:15


Exam format: exam will include short answer (few word to few sentence), problems, and drawing of certain structures or diagrams. I will ask you to APPLY the things we went over in class to new situations.

Study tips: review your notes, read the material I gave you to read, look over the questions at the ends of the Chapters in your text, and think, think, think!

Don't hesitate to email with questions.  You can also call me--email for phone #--I don't want to post here. 

Construction and Cloning of libraries

Terms to know:
Library
Partial digestion
DNA hybridization
Immunological Assay
Protein activity Assay
probe
Random priming
cDNA
genomic library
cDNA library
Klenow
reverse transcriptase
Rnase
S1 nuclease

Concepts to understand:
· Basic steps involved in creating a prokaryotic DNA library.  Be sure to know how to explain/illustrate this.  (review figure 4.12 and 4.13)
· What is the purpose of creating a library?
· How can you screen a DNA library?
· Know the basic method of screening by DNA hybridization, immunological assay or protein activity or functional complementation. Think about different protein activities you might be able to screen for. (review figure 4.14, 4.17and 4.18, 4.19 plus class notes)
· What are the sources of probes?
-How do you label probes? (review figure 4.15)
· Basic steps involved in creating a eukaryotic DNA library. Be sure to know how to explain/illustrate this.  How and Why is it different than creating a prokaryotic library?
What is the difference between a genomic library and a cDNA library?
How do you purify mRNA from total cellular RNA?(review figure 4.20)
How do you make a cDNA library?  How can you screen a cDNA library?(review figure 4.21a)
**Review Figure 4.22 which shows selecting and cloning full-length cDNAs

Expression of recombinant proteins in eukaryotic cells:


Terms to know:
Transfection
Calcium phosphate
Electroporation
lipofection
Microinjection
Post-translational modification
Thymidine kinase
G418
SV40
Vaccinia
Baculovirus
Retrovirus
LTR
Packaging cell line

Concepts to understand:

  • Why would you want to express a protein in a eukaryotic cell?
  • How can you get DNA into eukaryotic cells?
  • Review the thymidine kinase selection system (what is the limitation of this technique?)
  • Dominant selectable markers - see table 12-1. We spoke about APH gene/G418 selection in particular. How does G148 kill cells?
  • Stable versus transient expression.
  • Review the key features that make good eukaryotic expression vectors. (Figure 12-5, plus euk and prok ori)
  • SV40 viral vectors - review steps involved, what are limitations? (Figure 12-9, 12-10)
  • Review the vaccinia virus expression system (Figure 12-11)
  • Review the baculovirus virus expression system (Figure 12-12)
  • Review retroviral expression. (Figure 12-13) What are the advantages of this system? What are some applications?
  • Think about the differences between the different viral vectors and why one might be used over another.  What are the advantages and disadvantages?

Recombinant DNA Techniques -  PCR

Terms to know:

Primer
Probe
Thermostable polymerase
Nitrocellulose/nylon membrane

Concepts to understand:

  • What is necessary for a PCR reaction? (What must you include in the reaction)
  • Know how primers should be oriented for PCR.
  • Know how to calculate the annealing temperature of a primer.  
  • Know the temperature cycles in a PCR reaction, and what happens at each temperature.
  • Know how the repeated cycles lead to amplification of a DNA template.
  • Review applications of PCR -- obtaining more sample (DNA Fingerprinting, genetic testing, viral screening), creation of restriction enzyme sites for cloning.

Laboratory:
Review the 2 recombinant DNA labs and the spectrophotometry/PCR  lab.  Be sure to review the handouts and your lab reports. Some of things that you should review are listed below:

· Know how to prepare and run an agarose gel, and how DNA is separated on the gel.
· Know how to construct and use a standard curve for determining DNA fragment size. What s plotted?  On what?
· How is Calcium chloride transformation done?  How is transformation efficiency determined?
· How was the recombinant plasmid selected during the cloning lab? Why can this approach not always be used?
· Know how to determine the orientation of an insert within a vector by restriction enzyme analysis.
· Understand the principles and practice of PCR. What is included in a reaction, and how is the DNA amplified?  Review primer design. 
· How can you use a spectrophotometer to determine DNA concentration?  DNA purity?
· *Always worth reviewing your solution making.*