Team:Exeter/lab book/proto/4

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Protocol 4

Protocols | Single Gene Plasmids and Enzyme Characterisation | Showcasing Polysaccharide Production | The 3-Gene Inducible Plasmid

Operon Construction | Glycobase

New England BioLabs Biobrick Assembly Protocol

Digestion Protocol

Upstream digest		Downstream digest
Upstream Part Plasmid	500 ng	Upstream Part Plasmid	500 ng
EcoR1-HF	1 µl	Xba1	1 µl
Spe1	1 µl	Pst1	1 µl
10X NEBuffer2	5 µl	10X NEBuffer2	5 µl
100X BSA	0.5 µl	100X BSA	0.5 µl
H2O	to 50 µl	H2O	to 50 µl

Digest the Destination Plasmid with EcoR1-HF and Pst1: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdb, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

Destination Plasmid DNA	500 ng
EcoR1-HF	1 µl
Pst1	1 µl
10X NEBuffer2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Incubate all three restriction digest reactions at 37 °C for 10 minutes and then heat inactivate at 80 °C for 20 minutes.

Ligation Protocol

Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.

Upstream Part digestion	2 µl
Downstream Part digestion	2 µl
Destination Plasmid digestion	2 µl
10X T4 DNA ligase Buffer	2 µl
T4 DNA ligase	1 µl
H2O	11 µl

Incubate at room temperature for 10 minutes and then heat inactivate at 80 °C for 20 minutes.

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