Team:SDU-Denmark/labwork/Protocols/3A

3A-Assembly -Ligation of 2 parts into a vector

1. PCR step; see PCR protocol
2. Digestion
BSA is only needed for conventional restriction enzymes, NOT fast digest
1. Digest step for Plasmid Backbone

if sample comes from miniprep
Enzyme Master Mix for Plasmid Backbone (25ul total)
• 5 ul Fastdigest greenbuffer / NEB Buffer 2
• 0.5 ul BSA
• 0.5 ul EcoRI-H
• 0.5 ul PstI
• 19 ul dH20 (0,5 ul less when using BSA)

If sample comes from PCR
Enzyme Master Mix for Plasmid Backbone (25ul total)
• 5 ul Fastdigest greenbuffer / NEB Buffer
• 0.5 ul BSA
• 0.5 ul EcoRI-HF
• 0.5 ul PstI
• 0.5 ul DpnI (Used to digest any template DNA from productio
• 19 ul dH20 (0,5 ul less when using BSA

Digest Plasmid Backbone
• Add 4 ul linearized plasmid backbone (25ng/ul for 100ng tota
• Add 4 ul of Enzyme Master Mix
• Digest 37C/30 min, heat kill Restriction Enzyme 80C/20 mi


2. Digest step for Insert X:

If sample comes from miniprep
Enzyme Master Mix for Plasmid Backbone (25ul tota
• 5 ul Fastdigest greenbuffer / NEB Buffer
• 0.5 ul BSA
• 0.5 ul EcoRI-HF
• 0.5 ul PstI
• 19 ul dH20 (0,5 ul less when using BSA