Template:SBB10 ConstructionFiles PaulinaT sbb21
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Construction of FokI+ cleavage domain basic part sbb21
PCR fk0001/fk0003 on FokI (505 bp, gp = A)
PCR fk0004/fk0002 on FokI (141 bp, gp = B)
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PCR fk0001/fk0002 on A+B (620 bp, EcoRI/BamHI)
Digest pBjk2741-Bca1144 (EcoRI/BamHI, 2170+910, L)
Product is pBjk2741-ssb21 {<FokI+>}
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fk0001 Forward EcoRI and BglII for FokI ccaaaGAATTCgtccAGATCTCAGCTGGTTaaatctgaactggaggag
fk0004 (F)Base Substitution FokI ccataaaaccaatTgcaatggcgccg
fk0003 (R)Base Substitution FokI cggcgccattgcAattggttttatgg
fk0002 Reverse BamHI for FokI gctagGGATCCaaaattgatctccccattgttg
JCA Notes
- Correct assembly, prefix should be ATG not GTCC
- Explicitly put in the name of the template plasmid, not just a description; ie, what the tube will have written on the cap
<pre> Construction of FokI+ cleavage domain basic part sbb21 PCR fk0001/fk0003 on K243000 (504 bp, gp = A) PCR fk0004/fk0002 on K243000 (141 bp, gp = B) --------------------------------- PCR fk0001/fk0002 on A+B (619 bp, EcoRI/BamHI) Digest pBjk2741-Bca1144 (EcoRI/BamHI, 2170+910, L) Product is pBjk2741-ssb21 {<FokI+>} --------------------------------- fk0001 Forward EcoRI and BglII for FokI ccaaaGAATTCatgAGATCTCAGCTGGTTaaatctgaactggaggag fk0004 (F)Base Substitution FokI ccataaaaccaatTgcaatggcgccg fk0003 (R)Base Substitution FokI cggcgccattgcAattggttttatgg fk0002 Reverse BamHI for FokI gctagGGATCCaaaattgatctccccattgttg
