Tk:robot-assembly

From OpenWetWare

Jump to: navigation, search

Protocols designed for 6 ul minimum pipet volume and 30 ul residual liquid in the tube

Input part DNA: 44.3 ng/ul

Digestion: Add 10 ul of plasmid to 50 ul of digest mix:

    • 29.5 ul enzyme 1
    • 29.5 ul enzyme 2
    • 29.5 ul BSA
    • 148 ul NEB Buffer 2
    • 994 ul water
  • digest 1 hour at 37, heat kill 20 minutes at 80

Use 15 ul of the digestion to load a robotically loaded 48 well E-Gel plate

Mix 7 ul of digested part 1 Mix 7 ul of digested part 2 with 13 ul of construction plasmid backbone at 3.2 ng/ul Add 13 ul ligation master mix

    • 52.6 ul T4 DNA ligase
    • 10.5 ul 1 M ATP
    • 68.4 ul T4 DNA ligase bufer
    • 210 ul water

Ligate 30 minutes at room temperature Heat kill 20 minutes at 80C

Add 66 ul of CCMB80 buffer to 6 ul of the ligation mixture

Transform 50 ul of cells with 6 ul of the mixture

Add 200 ul of SOC, grow out for 2 hours at 37C

Serial dilute 1 part into 2 parts (3x dilution) across 12 wells into LB + antibiotic + indicator (Cresol Red)

Grow overnight. Select the last well showing growth.

Personal tools