Smith-ProteinExperession

Question 1

What are the four types of PCR we discussed?

Answer 1

1. Normal PCR
2. Overlapping PCR
3. Quickchange
4. TAQ/PFU

Question 2

Draw out what happens during normal PCR.

Answer 2

Smith-2 slide 7

Question 3

Draw out what happens during overlapping PCR

Answer 3

Smith-2 slide 8

Question 4

What are the problems with blunt end ligation? (3)

Answer 4

1. religating empty vector
2. GOI insertion orientation can be flipped
3. low efficiency

Question 5

Draw out what happens during quick change, mutagenesis PCR.

Answer 5

Smith 2 - slide 15

Question 6

What are some common protein expression problems you can run into?

Answer 6

1. Rare codons
2. Inclusion bodies

Question 7

What models can you use for protein expression?

Answer 7

1. E.coli
2. Yeast
3. Baculovirus
4. Lentiviruses
5. Animal Cells
6. Plant Cells

Question 8

What are the cons of using E.coli for protein expression?

Answer 8

1. can form inclusion bodies
2. issues with protein disulfides
3. no glycosylation (or other eukaryotic post-translational modification)

Question 9

What are the cons for using yeast cells for protein expression?

Answer 9

1. slower to grow and transform than bacteria
2. harder to crack open the cells than bacteria
3. more expensive to label than bacteria
4. not as many vectors and tools

Question 10

What are the pros and cons of using baculoviruses for protein expression?

Answer 10

Pros:
1. better post-translational modification system
2. better folding of mammalian proteins
Cons:
1. very expensive (media, plasticware, etc.)
2. ~2weeks for expression