FAST-Licase Protocol

FAST-Licase – Quick Protocol

Design Homologous Overlaps

Length

  • 15 bp to 20 bp.

  • 30 bp to 40 bp for inserts > 5 kb, more than two inserts, or for mutagenesis over large areas.

Sequence

  • Taken on either end or shared between the ends of the two DNA fragments.

  • For novel DNA insertions, mutagenesis or other engineering applications: de novo sequence is added at both ends of the DNA fragments and participates to the overlap.

Structure

  • No stable hairpin.

  • No DNA repeats.

Prepare DNA Fragments

PCR Fragments

  1. Create an in silico template sequence and design primers.

  2. Amplify DNA fragments by PCR.

  3. Check amplification by gel electrophoresis.

  4. Treat PCR reaction by DpnI (1 µl per 50 µl reaction, 1 h to overnight at 37°C).

  5. Optional: Gel-purify the desired PCR product.

  6. Purify the DNA fragment using spin column and elute in a small volume (< 50 µl) of pure water.

  7. Measure DNA concentration by UV spectrophotometry.

Restriction Digested Fragments

  1. Digest the plasmid DNA with restriction enzymes.

  2. Optional: Heat-inactivate restriction enzymes.

  3. Optional: Gel-purify the cut vector DNA. Background may be increased if this step is omitted.

  4. Purify the DNA fragment on a spin column and elute in a small volume (< 50 µl) of pure water.

  5. Measure DNA concentration by UV spectrophotometry.

Synthetic DNA Fragments

  1. Resuspend dry synthetic DNA fragment in pure water, at appropriate concentrations, such as 5 ng/µl.

Set Up the FAST-Licase™ Reaction

Calculate DNA amounts

  1. Choose the amount of vector

By convention, the vector is the DNA fragment that contains the origin of replication. Use the following table to determine the amount of vector to add to the reaction:

Vector Length

DNA Amount

< 4,000 bp

 50 ng

4,000 – 6,000 bp

 75 ng

6000 – 10,000 bp

 100 ng

> 10,000 bp

 150 ng
  1. Choose the insert to vector molar ratio

With a vector linearized by restriction enzymes, use a insert/vector molar ratio of 2. If the vector was amplified by PCR, use a ratio of 1. For insertion of small DNA fragments, use a ratio of 4 (< 400 bp), 6 (< 300 bp) or 10 (< 200 bp).

  1. Calculate the amount of insert

insert (ng) = vector (ng) x [insert length/vector length] x ratio

Run the FAST-Licase™ Reaction

Reagent

Amount / Volume
Pure water To 20 µl final volume
5x buffer 4 µl
Vector DNA As calculated
Insert DNA As calculated
Licase™ complex 1 µl

All reagents must be added successively in a single tube on ice to a final volume of 20 µl. The licase™ is added last. Mix briefly; hold the tube 5 s in a water bath set to 75°C. Keep on ice or at room temperature on the bench before bacterial transformation. For long term storage, keep at -20°C.

Analyze Transformants

Transform bacteria

Follow the manufacturer’s recommendations for the transformation protocol. Use chemically competent cells with medium efficiency in the range of 1-5 x 108 cfu/µg DNA. Mix 1 µl of the FAST-Licase™ reaction per 25 µl of cells. The volume of SOC can be limited to 200 µl per reaction.

Analyze clones

Pick 4 colonies and grow overnight at 37°C with agitation at 250 rpm in 3 ml of 2xYT medium supplemented with the proper antibiotic in 14 ml Falcon® tubes or equivalents.

The next day, spin all 4 tubes at 5000 rpm for 10 min and discard the supernatant. Freeze two tubes at -20°C for backup. Prepare the plasmids from the two other tubes and analyze the DNA for proper assembly.

 

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