Tk:offset cutters

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  • BspQI, SapI: gctcttc 1/4 HK, no meth, exp/200/BspQI exp/50 SapI digest 50 for BspqI
  • EarI: ctcttc 1/4 HK65, no meth, exp/500 100/100/50/100


  • BspMI: acctgc 4/8 HK65, no meth, exp/100, Buffer 3 only 0/0/100/0
  • BfuAI: acctgc 4/8 HK65, no meth, exp/250 50 deg/ 50% active at 37 0/75/100/10 Blocked by EcoKI methylation; A*CCnnnnnnGTGC; do not use tgcn as overlap
  • AarI: cacctgc 4/8 HK65, no meth, buffer pH 6.5 100 mM KCl 10 mM Bis-Tris-Propane 10 mM MgCl2, 500 nM oligo substrate
  • BsmBI: cgtctc 1/5 HK80, no meth, exp/200 75/100/100/100


  • BbsI: gaagac 2/6 HK65, no meth, exp/200, store at -70 100/100/25/75


  • BsaI: ggtctc 1/5 HK65, dcm blocked, exp/1000, 75/75/100/50 50 deg/ 10% active at 37 Blocked by dcm methyation, do not use CCWGGTCTC as site


Choose BfuAI and BsaI as cheap, both active at 50C, HK, buffer 3


Recutters:

  • BfuAI: potential sites: ggtACCTGCnnnnXXXX (Acc65I, ggtacc) or ACCTGCgcanXXXX (FspI, tgcgca)
  • BsaI: potential sites: accGGTCTCnnnnXXXX (AgeI, accggt) or nnnGGTCTCgagnXXXX (initial nnn not CCW) (XhoI, ctcgag)
  • BEAD : 3' biotin : PCR tag : BsaI cut site : XXXX PART1 XXXX : PstI cut site : AarI cut site(rev) : PCR Tag : EcoRI cut end


Plasmid:


  • Cut plasmid with AarI and EcoRI
  • Cut bead with AarI
    • wash beads in buffer + oligo
    • wash beads in buffer + enzyme
    • digest at 37°C 1 hour
    • wash ligase buffer
    • heat kill 20 minutes/65°C
    • wash ligase buffer
  • Ligate plasmid product (excess) to bead
    • Mix ligase buffer + ligase + new part
    • load ligation mix
    • incubate at 37°C
    • wash NEB buffer 2
    • heat kill 20 min 65°C
    • wash NEB buffer 2
  • capping reaction: cut with PstI, Klenow with dNTPs or exonuclease
    • wash with capping mix
    • incubate 20 min 37°C
    • wash
    • heat kill 20 min 65°C


  • Takara Biotech T4 ligase data sheet says AGCT sites (HindIII) are easiest to ligate, followed by PstI sites.
  • Choose ggAGCA or ggTGCT as the offset cut site, both coding for gly-ala


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