Library screening

Question 1

What is the first step in screening a DNA library?

Answer 1

Identification of the E. coli plaques that contain the DNA sequence of interest

Question 2

What are the 2 different ways the DNA sequence can be identified?

Answer 2

The pure sequence itself

The gene product- proteins or enzyme activity

Question 3

Explain how an amylase gene could be screened for

Answer 3

Plate the library on a starch-containing agar plate
Incubate overnight and flood the plate with I2 and KI \
The clone containing amylase will have a clear ZOI where starch digestion has occurred

Question 4

Describe antibody screening

Answer 4

Purify the protein of interest
Raise antibodies to that protein by injecting into rabbit
Plate the library on agar plate
Transfer library to filter
Lyse cells to release the proteins
Add the inert protein (usually bovine serum albumin) to block any non-specific binding
Incubate with radioactive antibody
Expose to X-ray film and radioactive antibody will blacken the film

Question 5

Describe DNA hybridisation screening

Answer 5

Plate library on agar 
Transfer to filter 
Lyse cells and denature DNA with alkali
Add salmon sperm DNA to block non-specific binding sites
Incubate with radioactive probe 
Expose to X-ray film

Question 6

What is DNA hybridisation screening used for?

Answer 6

cDNA or genomic libraries

Question 7

Where can DNA hybridisation screening take place?

Answer 7

In solution

With target DNA attached to nylon or nitrocellulose filter

Question 8

Describe differential screening?

Answer 8

Make cDNA library from liver
Hybridise with labelled total cDNA from liver and muscle
Clones hybridising only to liver cDNA are differentially expressed