Richard Lab:Amplified insert assembly

From OpenWetWare
Jump to navigationJump to search

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. A You-Tube video summary of the Amplified Insert Assembly method provides a quick and entertaining visual introduction.

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.

Notes

  • The DpnI eliminates any background from the insert PCR.
  • The phosphatase eliminates any background vector.
  • The "vector" will be digested for a total of thee hours (including nearly one hour with Antarctic Phosphatase)
  • The "insert" will only be digested for one hour. This is okay as there is a lot of it.
  • Detractors of this method may say that it's risky to PCR the inserts because of mutations. We say:
  1. This hasn't been a problem for us.
  2. This is why we use a high-fidelity polymerase
  3. We're sequencing the constructs anyway so we'd spot any mutations.

References

Contact

Back to Protocols

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Richard_Lab:Amplified_insert_assembly&oldid=598810"