From 2007.igem.org

DGAT biobrick: coR1-Pst1 digestion

The double digestion allows us to insert the biobrick into biobrick plasmid.
The Pst1 digestion allows us to see if the mutagenesis worked (dgat gene not digested).

• Material:

- DNA (PCR product DGAT deltaPst-1 biobrick from August 2): 200ng = 20µL
- BSA: 0.5µL
- NEBuffer EcoR1: 5µL
- EcoR1 enzyme: 1µL
- Pst-1 enzyme: 1µL
- ddH₂O

• Protocol:

- 2 hours at 37°C

• Purification

Quantity:

TOPO cloning

Cloning using TOPO protocol of the following purification of PCR products

Number	Name	Oligo F	Oligo R	Matrix	Length	Comments
P6	Lox66-DapASubtilis	O8 Lox66-DapASubt-F	O9 DapASubt-R	B.Subtilis 168	979
P7	Lox71-FtsZ	O1 Lox71-FtsZ-F	O2 FtsZ-R	MG1655	1260	EcoRI site in FtsZ not mutated
P8	RBS-DapAColi	O20 RBS-DapAColi-F	O7 DapAColi-R	MG1655	952	B0030-DapAColi