IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/29

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pBBR1MCS-5 Double digestion

Abstract

I made a double digestion with PstI and EcoRI of the plasmid pBBR1MCS-5 that Paulina extracted.

I need this plasmid digested to ligate all the anderson collection promoters.

Details

I do not exctly know the plasmid pBBR1MCS-5 concentration or even if the extraction was good, but Paulina says it was, I have to ask her for the gel image.

I made 4 reactions, 3 were from 3 different pBBR1MCS-5 extractions or samples and one was as a control from a part that Fabricio had already made and went well, this part has a RFP.

Reactive amounts and details

Reactive Amount (μL)
MiliQ Water 5
10X Buffer H invitrogen™ 3
template (pBBR/ctrl) 20
EcoRI (invitrogen™) 1.5
PstI (invitrogen™) 1.5
Total 31
  • I left the reaction incubating at 37°C at 6:14p.m., Fabricio will take it out when it is ready.
  • The enzimes should be inacticvated at 80°C for 20 minutes (check the manuals in the reactive amounts table)



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