IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2014/08/06

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6th August 2014

  • Set up level 2 dig-lig reactions (Vector, buffer, inserts, ligases)

Level 2 Constructs:

A= 35s_AvrBS3 + BS3_GFP

B= 35s_AvrBS3 + BS3_NbHb1

C= BS3_Bax + BS3_Bi1RNAi

D= 35s_AvrBS3 + BS3_Bax +BS3_BiRNAi

E= 35s_Bax1 + 35s_BiRNAi

F= PDF1.2_Bax1 + PDF1.2_BiRNAi

G=PR1_Bax + PR1_Bi1RNAi

  • PCR of Level 2 Constructs (cycles: 3 cycles of
 10 minutes 37°C,
 10 minutes 16°C, 
followed by 
10 minutes 37°C, 
20 minutes 65°C
, 4°C forever)
  • Made 1µl per ml Streptomycin plates. Spread with 100µl IPTG and 80µl of X-gal per plate
  • Transformed 5µl of level 2 construct DNA from PCR into 50µl E.coli cells, electroporation of cells, spread (amount?) onto streptomycin plates, incubated overnight at 37oC