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. We recommend using the following protocols.

Restriction Digest Protocol

estimated time: 30 min. active, 50 min. incubation

The following protocol assumes you'll be doing restriction digests for 3A assembly, therefore we refer to your digests as:

• Part A (The 1st part in the future composite part)
• Part B (The 2nd part in the future composite part)
• Linearized plasmid backbone (The destination plasmid backbone for your composite part)

If you are simply doing a restriction digest for quality control, you can use the protocol below.

Restriction Digest from iGEM Videos.

Materials

• Ice and bucket/container
• (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
• Part A (Purified DNA, > 16ng/ul)
• Part B (Purified DNA, > 16ng/ul)
• Linearized plasmid backbone (25ng/ul)
• dH2O
• NEB Buffer 2
• BSA
• Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
• Thermal cycler

Notes:

• You should keep all materials on ice.
• At iGEM HQ we use restriction enzymes from New England Biolabs

Single Reaction

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.
   
3. Add 0.5ul of BSA.
   
4. Add 0.5ul of EcoRI.
   
5. Add 0.5ul of PstI.
   
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 (8ul, 100ng), to check that both plasmid backbone and part length are accurate.