• pExpression plasmid sample (eg. pAD-ORF) or PCR product with attB sequencing flanking the desired gene fragment.
• pDONR plasmid sample (pDONR221, pDONR223, etc.)
• BP clonase mix II (5x)
• 96-well PCR plate x 2
• SOC
• NEB 5-alpha competent cells

• Prepare 6-60ng/ul solution of attB PCR sample/ plasmids.

• Prepare 2 96-well PCR plates indicating name, sample, and date.
• Add 2ul of the DNA as follows into one PCR plate.

• Prepare Ice with a tube labled “BP”
• Thaw BP clonase mix II on ice (~2mins).
• Breifly vortex (1sec x 2), and centrifuge.
• Take Xul of clonase mix from tube and aliquot to the labled tube.

• Return enzyme mix to -80C freezer.

• Add X µl of ddH2O and pDONR plasmid to the 5x BP clonase mix to make a 2x BP clonase master mix.
• Pipette well to mix the enzyme mix until the solution is completely mixed.
• Add 2 µl of the 2x enzyme mix to each of the the DNA mix prepared in PCR plate.
• Incubate @25C on a PCR machine O/N (>16h)

• Prepare the reaction mix and competent cells on ice.
• For each sample, add 10µl of homebrew competent NEB5-α cells.
• Incubate on ice for 20mins
• Heatshock on PCR machine @42C for 30secs
• Incubate on ice for 2mins
• Outgrowth by adding 100ul S.O.C. and incubate @37C for 1 hour (static)
• Spot 5 µL of each sample to LB+Amp plates
• Culture/select the transformats by incubating the plate, @37C, O/N.

— Dan Yamamoto-Evans 2019/11/18 17:46