Help:Protocols/Restriction Digest

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Restriction Digests

When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest.

At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for

Materials

• PCR tube
• dH20
• Enzymes (EcoRI, XbaI, SpeI, PstI)
• BSA
• Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.

Procedure

1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
   
2. Add 2.5ul of NEBuffer 2 to the tube.
   
3. Add 0.5ul of BSA to the tube.
   
4. Add 0.5ul of your first enzyme.
   
5. Add 0.5ul of your second enzyme.
   
6. There should be a total volume of 20ul. Mix well and spin down briefly.
   
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
   We incubate in a thermal cycler with a heated lid
   
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. Use ~2ul of the digest (20ng of DNA) for ligations.

3A Assembly Procedure

	Part A	Part B	linearized plasmid backbone
DNA	20ul	250ng	250ng	250ng
NEB Buffer 2	2.5ul	2.5ul	2.5ul
BSA	0.25ul	0.25ul	0.25ul
Enzyme 1	0.5ul EcoRI	0.5ul XbaI	0.5ul EcoRI
Enzyme 2	0.5ul SpeI	0.5ul PstI	0.5ul Pst1