IGEM:University of Debrecen:Restriction biobrick parts

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Contents

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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Part A

Part B

Backbone

Nuclease free water (in μL)

75

75

75

Tango Buffer 10X (in μL)

10

10

10

Volume of DNA to be restricted (in  μL)                          (if in concentration of  1 μg/ 1 μL)

5

5

5

Restriction Enzyme 1 (in μL)

5

5

5

Restriction Enzyme 2 (in  μL)

5

5

5

Total (in  μL)

100

100

100

















Notes

The volume of restriction enzymes used must not exceed 10% of the reaction volume (enzymes are dissolved in glycerol which damages the reaction).

Before starting the reaction calculation it is vital to examine the enzymes information page in order to obtain:


Optimal working buffer -May cause a change in the procedure stated above

Optimal working temprature -May cause a change in the procedure stated above

Inactivation temperature -May cause a change in the procedure stated above


If you need a higher backbone concentration, try our PCR backbone protocol

If you need a higher parts concentration, you should probably do a maxiprep

References

1. Sean C. Sleight, Bryan A. Bartley, Jane A. Lieviant, and Herbert M. Sauro "In-Fusion BioBrick assembly and re-engineering" Nucleic Acids Res. 2010 May; 38(8): 2624–2636.

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