Cloning via DNA Assembly
DNA Assembly in a 1:2 ratio of vector to insert with gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using QuickCIP). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.
For Crtl, pCFD6 BbsI AP was wrongly used.
DNA Assembly in a 1:2 ratio of vector to insert with the gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using FastAP and 2 extraction steps). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.
For Crtl, pCFD6 BbsI AP was used in reaction.
Success: rsh Stock has rsh1 Mutation
Via gDNA analysis and PCR was the specific area of the rsh gene extracted and amplified where the nucleotide substitution: C to T (Folkers et al., 2006) should be for the rsh1 mutation. The amplicon was Sanger sequenced which proved the nucleotide substitution.
Creating gRNAs via PCR
The template pCFD6 was used with a concentration of 640 pg/µl.
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
The template pCFD6 was used with a concentration of 128 pg/µl (1:5 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
The template pCFD6 was used with a concentration of 640 pg/µl.
The Phusion DNA Polymerase was used instead of the Q5 High-Fidelity DNA Polymerase.