Tk:offset cutters

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

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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