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


 * 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


 * Sherline
 * Biohit rLine pipettor http://www.biohit.com/view/products.asp?document_id=311&cat_id=276
 * Millipore hydrophilic ptfe membranes
 * NEB biotin beads: 1 micron, 4 mg/ml, 1 nM/mg beads
 * typical plasmid 5000 bp, 100 ng, 5e3*660 g/mole = 3e6 g/mole
 * for 100 ng plasmid, we have 1e-7 g / 3e6 g/mole = .3e-13 = .03 pM
 * Seiko D-Tran
 * http://www.homeshopcnc.com/index.html
 * http://www.kdntool.com/_sgg/f10000.htm
 * biofluidix.com
 * drddiluter.com 978 210 6390; 978 590 5907
 * http://www.nichiryo.co.jp/en/products/custom_made/pipettes.html
 * lab in a tip
 * http://www.hudsoncontrol.com/products.html
 * http://glygen.com (streptavidin coated tips)
 * shop sabre xyz tables
 * http://www.cnc-step.com
 * http://www.hsi-inc.com/linear_actuators.php
 * http://www.nookindustries.com/
 * http://www.imshome.com/mdrive23plus_mdi.html
 * http://www.techno-isel.com/Tic/LinMoProducts.htm
 * http://www.vxb.com/page/bearings/CTGY/LM?gclid=COHWoZShq5ACFQwsOAodGALDNw
 * fab at home
 * candyfab.org
 * Tecan/Cavro (oem) syringe pumps
 * Hamilton OEM syringe pumps
 * LM Biotech Xiril
 * mcwire cnc
 * reprap
 * http://www.kdntool.com/_sgg/f10000.htm