GGA for BsmBI

We are trying to clone two gBlocks designed with sticky ends after digestion with BsmBI.

Thermocycle Program

• 20 minutes at 55 C (start with digestion cocktail below)
• 5 minutes at 16 C
• push pause when it reaches 16 C
• put in 5 µL ligation mixture (see below)
• push pause again to resume 5 minute timer
• (when 5 minute timer is done, start loop below)
  • 2 min 16 C
  • 1 min 55 C
  • (end loop) 20 cycles total
• hold at 16 C

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Digestion cocktail

• • water 6.5 µL
  • 25 ng vector cut with EcoRI and Spe (gel purified) (1.76 µL)
  • 3x molar insert 1 (P1) 1 µL
  • 3x molar insert 2 (P2) 1 µL
  • 10x cutsmart buffer 1 µL
  • BsmBI enzyme 0.5 µL
• final volume 10 µL

Ligation mixture

• • water 3 µL
  • 10X ligation buffer 1.5 µL
  • ligase 0.5 µL