IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/04

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Promoters Digestion

Abstract

I digested all the 20 anderson promoters with EcoRI ans PstI (bouble digestion) so we can ligate them later with the plasmid pBBR1MCS5 backbone

Details

In total I made 20 reactions, 1 for each promoter (J23100-J23119)

Reactive ammounts

Reactive Ammount (μL)
MiliQ Water 27
Buffer (EcoRI) 5
BSA 1
Template DNA 15
EcoRI (NEB™) 1
PstI (NEB™) 1
Total 50

Process details

  • I left the reactions overnight (8.5 hrs) at 37°C
  • I inactivate the enzymes 80°C for 20 min which is the inactivation conditions for PstI. EcoRI inactivation temperature is 65°C minimum.

Results

There is not an easy way to know if the digestions were well until I do the ligation step.