Richard Lab:Amplified insert assembly

Back to Protocols Amplified Insert Assembly

Overview
This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the consensus protocol.

This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:

-EcoRI--XbaI--Part--SpeI--PstI-

Ocassionally other enzymes (e.g. BamHI or HindIII) are used to make protein fusions. See our bio-brick format page for more details.

The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids. The eventual goal of assembly is to get these parts on the same plasmid next to one another.

Procedure
1. Miniprep both "insert" and "vector" from their respective cultures using a kit or this protocol (30 mins). 2. PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling).
 * Use a high-fidelity polymerase (e.g. pfu Turbo or Vent).
 * Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).
 * Only run 25-30 cycles as this will help ensure high fidelity.

3. Start Digesting the "vector" while the PCR is cycling.
 * For help on deciding which enzymes to use see this page.

4. Purify the PCR product using a kit or this protocol. 5. Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases). 6. Once the insert is digesting add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. 7. Kill all reactions by incubating for 20 mins at 80°C. 8. Ligate at a molar ratio of 4:1 (insert:vector). 9. Transform. 10. Plate on plates with the same antibiotic as the "vector" resistance. 11. Celebrate.


 * If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours.

Contact

 * This protocol was developed by Mike Speer and Dr. Tom Richard
 * You can also discuss this protocol here.

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