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)
---------------------------------
PCR fk0001/fk0002 on A+B           (620 bp, EcoRI/BamHI)
Digest pBjk2741-Bca1144            (EcoRI/BamHI, 2170+910, L)
Product is pBjk2741-ssb21          {<FokI+>}
---------------------------------
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
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