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Flashcards in Library screening Deck (8)
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What is the first step in screening a DNA library?

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


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

The pure sequence itself
The gene product- proteins or enzyme activity


Explain how an amylase gene could be screened for

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


Describe antibody screening

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


Describe DNA hybridisation screening

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


What is DNA hybridisation screening used for?

cDNA or genomic libraries


Where can DNA hybridisation screening take place?

In solution
With target DNA attached to nylon or nitrocellulose filter


Describe differential screening?

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