Gateway Cloning

Materials and Reagents

BP reactions

  • 2 μL of pDonr221 (150 ng/μL) in T.E.

  • 60 ng of attB PCR product

  • 2 μL of BP clonase

  • 5 uL of T.E.

LR reactions

  • 2 μL of Destination Vector (150 ng/μL)

  • 200 ng/μL of pDonr221 library

Procedure

For electroporation of library

  1. Combine two 10 μL LR reactions.

  2. Remove salts through ethanol precipitation.

  3. Resuspend into 2.5 μL of T.E.

BP reactions

  • Incubate for 18 hours at 25 degrees Celsius.

  • The source document notes that this gives around 600,000 clones.

Notes and Cautions

  • BP and LR clonase have different optimal conditions. For optimal efficiency, necessary to perform Proteinase k digestion (>4x improvement).

  • If the initial library is made by recombining into pDonr221 and then maxi-prepping the library, one should perform EcorV digestion of the pDonr221-library prior to the LR reaction.

  • Combining two reactions for every 50 μL of electrocompetent cells appears to work well. No additional gain observed when combining four reactions.

  • The LR reaction works best if the donor fragment has been relaxed. So long as your gene does not contain EcorV, this restriction endonuclease works well at site-specifically cleaving the vector backbone of pDonr221.

  • Alternatively, the gene to be cloned into your destination vector with LR Clonase II can be amplified from a pDonr221 parent with M13Forward and M13Reverse.

  • The latter is ideal for generating libraries since this decreases cost, preserves diversity of the library, and saves time.