IGEM:University of Debrecen:Restriction biobrick parts

Scientific Background
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.

In the process of chaining biobrick parts together, the restriction sites between the two parts are removed, allowing the use of those restriction enzymes without breaking the new, larger BioBrick apart.[4] To facilitate this assembly process, the BioBrick part itself should not contain any of these restriction sites.[1]

One such type of assemblies is the “three antibiotic” assembly standard. This assembly begins with a restriction step.

Overview
The following protocol contains detailed instructions on the restriction digestion step of the “three antibiotic” standard assembly. It starts with a medium amount of the two parts to be assembled and a medium quantity of the backbone that the parts will be assembled into. The result is a small amount of the insert part ready to be ligated into a PCR amplified backbone.

Note: This protocol uses Fermentas restriction enzymes and buffers

Note: One unit of restriction enzyme cuts 1ug of DNA in 1 hour.

Procedure
1. Prepare the following mixes in 3 different PCR strips, work on ice.

Note: the restriction of Part A, Part B and the backbone at the same reaction is not mandatory and may be split over several reactions

2. Resuspend with a pipette all the components to assure proper mixing

3. Start the following thermocycler program:

37°C for 4 hours (see notes)

80°C for 20 minutes

4°C forever

4. Run products on gel electrophoresis. (Nice bands on gel are made by 300-500ng of DNA, thus use 8 ul of the reaction volume at least for gel)

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