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From 2010.igem.org

Construction plasmid is uncut backbone plasmid DNA from the three different plasmid backbones used for three antibiotic assembly (pSB1C3, pSB1K3, and pSB1T3). Once it is cut with EcoRI and PstI it is ready for use in three way ligation reactions. Ideally, it is free of inserts and circular DNA. There are several ways to make construction plasmids. Our original approach was to use plasmid containing the P1010 insert, coding for the ccdB protein generator. This protein product is toxic to cells not having the gyrA462 mutation (most strains except for DB3.1). As a result, uncut or religated plasmid would produce little background. In our experience, this approach has a number of difficulties. Extraction of cut backbone from inserts required the use of gel purification, which is difficult with high concentrations of DNA, and problematic in yield and quality. The P1010 insert also showed remarkable ability to mutate (usually by frame shift) to a form not toxic to normal cells, leading to significant background.

Our revised approach is to build construction plasmid using PCR techniques. Using primers which match the biobrick cloning sites in the reverse orientation, we produce PCR product containing the plasmid backbone with no insert and no uncut, circular plasmid. Errors are introduced in this PCR process, but the errors are of two forms: ones which result in plasmids which cannot be transformed, and those which produce viable, but mutated plasmid backbone. In either case, we don't care, since the insert sequence is what we care about.

The PCR product is cut with EcoRI and PstI, purified away from the short dsDNA fragments cut off, and used for assemblies. Since the quality of this construction plasmid is a key determiner in the success of three antibiotic cloning, substantial effort has been put into optimizing the reactions. This protocol describes the exact procedures and quality control techniques used to make the construction plasmid backbones.

Contents 1 PCR 1.1 Materials 1.2 PCR reaction 2 Purification 2.1 Qiagen purification option 3 Restriction enzyme digest

PCR

Materials

• Template DNA for the backbone of choice (any plasmid with the correct backbone) at 10 ng/μl concentration.
• Invitrogen PCR Supermix High Fidelity (Invitrogen 10790-020)
• Primer Suffix-F, sequence actagtagcggccgctgcag at 30 pmol/μl
• Primer Prefix-R, sequence tctagaagcggccgcgaattc at 30 pmol/μl

PCR reaction

Mix a PCR reaction consisting of:

• 300 μl PCR supermix
• 6 μl each primer
• 0.5 μl template DNA
• Aliquot into 100 μl samples for the cycler

Run this PCR program:

1. initial denaturing 95 C for 5 minutes
2. denature 94 C for 30 s
3. anneal 55 C for 30 s
4. extend 68 C for 4 minutes
5. cycle 36 times to step 2
6. final extension 68 C for 10 minutes
7. hold at 4 C indefinitely

Purification

Qiagen purification option

We have also used a Qiagen [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx?r=1745 PCR purification kit] to get rid of the enzymes in the PCR reaction. It is ideal to use the PCR product that has been Qiagen PCR purified immediately after purification.

Restriction enzyme digest

Once your plasmid backbone has been amplified and purified you must digest it with the flanking restriction enzymes that you used to cut your two parts that you are assembling. In [http://partsregistry.org/partsdb/scars.cgi assembly standard #10] (BioBrick standard assembly) you would use EcoRI and PstI.

You can use the digested product directly in the ligation after having digested (@37 degrees) and heat killing (@80 degrees). It is best to use it soon after the reaction has completed.

Alternatively you can do another purification and store the product at -20 degrees.

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