Tk:robot-assembly: Difference between revisions
(New page: 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 enzy...) |
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Latest revision as of 19:30, 7 November 2008
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.