Principles Of Cloning Flashcards Preview

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Flashcards in Principles Of Cloning Deck (20):
0

Methods of transfer of genetic material

- conjugation
- transduction
- transformation

1

Why do we clone?

- reproduction if specific DNA fragments in large amounts
- stable propagation of DNA sequences
- allows DNA to be studied, manipulated and expressed

2

Applications of cloning

- construction of genetic libraries
- production of scarce proteins
- mass production of vaccines
- diagnostics
- therapeutics
- gene transfer

3

What is the restriction modification system.

Host DNA is methylated to protect it from cleavage by REs

4

What factors are Re groups based on?

- composition
- enzyme cofactor requirements
- nature if target sequence
- position of DNA cleavage sites relative to target sequence

5

How does DNA ligase work

Forms phosphodiester binds between 5' p and 3' OH

6

What is cDNA used for?

- expression studies
- gene structure and function studies

7

4 strategies for cloning PCR products

- blunt end PCR cloning
- restriction site cloning
- T/A cloning
- SDM

8

What must cloning vectors contain

- origin if replication
- selectable marker
- multiple cloning site

9

What are expression vectors

Allow a cloned segment if DNA to be translated into a protein inside a cell

10

What must expression vectors contain?

- in vivo promoter
- antibiotic selection
- sequencing primers

11

What is a linker.

Small dsDNA that contains a new RE site

12

What is directional cloning?

Cut both vector and insert with 2 different REs
Low background of non-recombinants

13

What does AP do?

Removes 5' phosphatase from vectors to prevent self-ligation
Decreases non-recombinants
NB in blunt-end cloning

14

How to calculate molar ratios

(Size of insert in kb/size of vector in kb) x ng of vector = ng of insert needed for a 1:1 ratio

15

Competency

The ability of a cell to take up extracellular naked DNA from its environment

16

How to increase competency

High ca2+ conc makes small holes in the cell membrane

17

Transformation efficiency

Number of colonies per ml/DNA conc transformed

18

Steps to isolate plasmid DNA

- re suspend in solution 1 (tris-HCl buffer, EDTA, Glucose)
- add solution 2 (SDS, Naoh)
- add solution 3 (potassium acetate, acetic acid)
- precipitate plasmid DNA with isopropanol
- restriction mapping

19

Types of plasmid DNA

Uncut
- nicked
- linear
- supercoiled
Cut
- linear