PCR pCDF6 Primer
-12.5 µl primer reverse/forward
-5 µl dNTPs
-2.5 µl Polymerase
-0.57 µl pCDF6 undigested
-50 µl Q5 buffer
-166.93 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction

pCDF6 NEBuilder Assembly Reaction
-pcr1: 4.85 ng -> 0.88 µl
-pcr2: 4.24 ng -> 1.5 µl
-pcr3: 4.87 ng -> 1.6 µl
-pCDF6 digested: 100 ng -> 2.8 µl
-H2O: 3.22 µl
-10 µl NEBuilder HiFi DNA Assembly Master Mix/Control
PCR
heat shock transformation of the construct into competent E.coli cells
-> plated on LB0+Amp plates
-> no colonies