IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/07

UIUC - Arsenic Collection System!

Main project page Previous entry      Next entry

Current Project Summary Part Status J23100 Minipreped J23119 Minipreped Gas Vesicles Minipreped ArsR TF ?PCRed? pArsR will PCR today LamB Need Primers ArsB Need Primers pRpoS Need Primers Also need primers for SDM of ArsR and Fusion of LamB & ArsR Constructs J23100 Gas Vesicles J23119 Gas Vesicles pRpoS Gas Vesicles pArsR Gas Vesicles J23100 ArsB J23119 ArsB J23100 ArsR TF J23119 ArsR TF LamB / ArsR with BsaI LamB / ArsR with BamH1 and KpnI LamB / ArsR with BamHI - more primers NOTE: need to order restriction enzymes: BsaI, but leave BamHI and KpnI until later 7/7/10 PCR Purified pArsR to 50 ng/ul. Only used 15 ul to purify, should have about 10 ul left in tube. Used purification product for digestion. Digestions pArsR DNA 10 ul (500ng) H2O 32.5 ul Buffer 2 5 ul BSA .5 ul SpeI 1 ul XbaI 1 ul pSB1T3 DNA 1 ul (500ng) H2O 41.5 ul Buffer 2 5 ul BSA .5 ul SpeI 1 ul XbaI 1 ul Incubate at 37°C 15 min. Inactivate at 80°C 20 min. PCR purified pSB1T3: No yield. PCR for pArsR 95 - 2min (95 - 30sec; 55ish - 30sec; 72 - 1min | 31x) 72 - 5 min 4 - hold Rxn Mixture Component ul 7x MM 10x Buffer 4 28 10mM dNTP 1t 7 Template (250ng/ul colony boil of E. Coli 2ul 5.6 10uM F Primer 4 -- 10uM R Primer 4 -- Pfu Turbo DNA Pol .4 2.8 H2O 24.6 172.2 Samples: 1 pArsR: F1 - 502 + R1 (540bp) 2 pArsR: F1-502 + R2 (547bp) 3 pArsR: F2 - 255 + R1 (293bp) 4 pArsR: F2 - 255 + R2 (300bp) 5 ArsR (380bp) 6 Neg Control - H20 instead of Primers Colony Boil: Swiped tip over Escherichia coli MG1655. Add 25ul dH2O Incubate at 98C for ~2minutes Yield was ~250ng/ul Gel of PCR Lane Sample Amount 1 1kb ladder 2ul 2-7 samples 1-6 5 ul sample, 1 ul dye 6 1 Kb Ladder 1 ul Run on 1% gel