Richard Lab:Amplified insert assembly

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Overview

This is what Mike calls "Standard Assembly 2.0" and is a method of "bio-bricking" two biological parts (i.e. pieces of DNA) together. For more information on bio-bricking see this link. This method combines the ease and speed of 3A assembly with the fidelity of standard assembly. Major benefits of this assembly method over other assembly methods include:

• no need for gel electrophoresis or gel extraction.
• the ability to insert small (i.e. invisible on a gel) parts.
• no need to fool with multiple antibiotic resistances.
• no having to make construction vectors.
• really low background transformation

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.

Procedure

Your two parts will be thusly labeled "insert" and "vector" and will be initially contained on separate plasmids. The basic steps are as follows:

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).

• Use a high-fidelity polymerase (e.g. pfu Turbo or Deep Vent).
• Use the same primers you use for colony PCR (Annealing Temp of 55°C).

1. Digest the "vector" for 2 hours.
2. Purify the PCR product using a kit or this protocol.
3. Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
4. Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.
5. Kill all reactions by incubating for 20 mins at 80°C.
6. Ligate (Don't forget to kill the ligation).
7. Transform.
8. Plate.
9. Celebrate.

This entire process should take around 6 hours as many steps overlap.

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 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:
  • This hasn't been a problem for us.
  • This is why we use a high-fidelity polymerase
  • We're sequencing the constructs anyway so we'd spot any mutations.

Contact

• Mike

or instead, discuss this protocol.

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