Gateway cloning Flashcards
(15 cards)
What does ‘att’ stand for in Gateway cloning?
Attachment site (from bacteriophage lambda).
What are the four types of att sites in Gateway cloning?
attB, attP, attL, attR.
What sites are involved in the BP reaction?
attB + attP → attL + attR.
What sites are involved in the LR reaction?
attL + attR → attB + attP.
What enzyme is used in the BP reaction?
BP Clonase™.
What enzyme is used in the LR reaction?
LR Clonase™.
What is the product of the BP reaction?
An entry clone (gene flanked by attL sites).
What is the product of the LR reaction?
An expression clone (gene ready for expression in destination vector).
What is the purpose of the entry clone?
To store your gene in a standardized format with attL sites for further use.
What is the purpose of the destination vector?
To receive your gene during the LR reaction and allow it to be expressed in cells.
What remains in the plasmid after recombination?
The recombined att sites (attB or attP).
Why must att sites be in the correct orientation?
To ensure proper recombination; wrong orientation = failed reaction.
How are attB sites added to a gene during cloning?
By including them in the 5’ ends of PCR primers.
What is the key equation to know?
Entry clone + Destnation vector - Expression clone + byproduct
