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I have left up last semester's review sheet as some students find these useful.  It will be updated for Spring 2010 2-5 days before the exam.

BIOT251 Review sheet Exam 3
Exam date: Friday April 24th, 2009 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.

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:
bulletWhy would you want to express a protein in a eukaryotic cell?
bulletHow can you get DNA into eukaryotic cells? Review the methods we spoke about in class
bulletReview the thymidine kinase selection system (what is the limitation of this technique?)
bulletSelectable markers - see table 12-1. We spoke about APH gene/G418 selection in particular. How does G148 kill cells?
bulletStable versus transient expression (Figure 12-4).
bulletReview the key features that make good eukaryotic expression vectors. (Figure 12-5)
bulletSV40 viral vectors - review steps involved, what are limitations? (Figure 12-9)
bulletReview the vaccinia virus expression system (Figure 12-11)
bulletReview the baculovirus virus expression system (Figure 12-12)
bulletReview retroviral expression. (Figure 6-12) What are the advantages of this system? What are some applications?
bulletThink about the differences between the different viral vectors and why one might be used over another.  What are the advantages and disadvantages?
bulletWhat are reasons you might choose one type of cell over another for expression (bacteria, yeast, insect, mammalian)
bulletWhat are GST and His Tags?  How can they help protein purification?