Colony PCR for screening colonies (CAT#: STEM-MB-0202-WXH)

Introduction

Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.<br />Traditionally, verification of DNA insertion during cloning was time-consuming, following bacterial transformation with laborious DNA purification and subsequent restriction enzyme digest.

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Principle Applications Procedure Materials Notes Related Products

Principle

The bacterial colony containing the plasmid can directly be amplified using two sets of primers.
The first set is of the insert specific primers which amplify the insertion sequence, and the other is of vector-specific flanking primers, which amplifies the plasmid DNA other than the inserted DNA.

Applications

Determining the presence or absence of insert DNA in plasmid constructs.

Procedure

1.Design primers to detect the presence of your insert;
There are 3 strategies for primer design: insert-specific primers, backbone-specific primers, orientation-specific primers
2.Set up a standard PCR reaction(primers, dNTPs, polymerase) using the supernatant of lysed bacteria as template;
3.Run PCR product on a gel to analyze product size

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