Lecture 11 Fluorescence Microscopy

Question 1

Lac Z expression present in vectors

Answer 1

Cloning vectors can encode a shortened derivative of Lac Z (Lac Z’)
The polylinker site (MCS) is located within the LacZ’ gene
Disruption of the lacZ in the vector leads to the white colonies and suggests an insert is present in the vector

Question 2

What colour are the colonies if there is no insert?

Answer 2

Blue colony if there is no insert in the vector - lacZ gene function normally (non-recombinant plasmid)
White colonies means there is an insert present since it disrupts the LacZ gene

Question 3

Preparation of Genomic Library vs Preparation of cDNA library

Answer 3

Genomic library
- Fragment of genomic DNA
- Various genomic sequences

cDNA library (complementary DNA)
- Takes out introns, join exons (splicing)
- Reverse transcription and then clone (mRNA -> cDNA)

Question 4

What is PCR?

Answer 4

Polymerase chain reaction
- Amplify a specific region of DNA and make numerous copies
- Thermostable polymerase (Taq Polymerase) which stays active at high temperatires
- Can be used to identify with a high probability, disease-causing viruses and or bacteria
- Specific primers must already know the exact sequences on both ends of a given reaction of interest in DNA (doesn’t need to know seq inbetween primers)
- Must design specific primers and determine the Temperature of melting (Tm) for each primer to know what temp for annealing of primer to template

Question 5

Types of PCR

Answer 5

1. Semiquantitative or end point - monitor at the end of 20-40 cycles
2. Quantitative PCR - real time PCR to find the actual amt of DNA you have
3. Reverse transcriptase PCR - to generate cDNA copies (the more RNA present, the more DNA is produced)

Question 6

What is chromatography? Prinicples?

Answer 6

Separate molecules on the basis of chemical properties (molecular mass, charge, solubility, affinity for a particular ligand or solid support)
Separate based on differential migration through a porous medium
Mixture of compounds have diff. affinities for the stationary phase (solid support or matrix) on which it is adsorbed and the mobile phase (buffer or solvent) passing through the stationary phase

Question 7

What are the types of chromatography/purification techniques?

Answer 7

1. Gel filtration
   - Separated based on size
   - Protein are washed with a buffer through a column
2. Ion-exchange
   - Based on charge of protein
   - If there is a positively charged gel beads, then the negatively charged proteins will be attracted to it and the positively charged proteins will elute out
3. Antibody-Affinity
   - Based on if protein is recognized by antibody
   - If the protein recognizes it, then it will be attached to the antibody and the result of the proteins will elute out
   - In general, it achieves a higher purification factor

Question 8

Reporter proteins

Answer 8

Easily detected and is not present normally in research system
They are used as “markers” for analysis in gene regulation and localization (analysis of mutation altered genes)
Beta-galactosidase is an example of reporter proteins

Question 9

What are transgenic organisms?

Answer 9

An organism that has had a modification to their genome and passed on to the next generation
Transgene: gene modified/added