title: UofT KB1 19.2K v2
status: Public on Jan 27 2010
contact.city: Toronto
contact.first: Elizabeth
contact.middle: A
contact.last: Edwards
contact.email: elizabeth.edwards@utoronto.ca
contact.address: 200 College st.
contact.department: Department of Chemical Engineering and Applied Chemistry
contact.country: Canada
contact.institute: University of toronto
organism: dechlorinating enrichment culture KB-1
description: KB1 mixed microbial consortium.  Composition of community as estimated by 16S qPCR:
  
  Dehalococcoides:48.1-70.2%
  Chlorobi SJA-28:6-14.9%
  Acetobacterium:2.65-12%
  Sporomusa:1.2-11.45%
  Spirochaete SA-8 :2.8-8.3%
  Methanomicrobiales:3.5-5.8%
  OP5:2.5-5%
  Geobacter:3-4.4%
  Methanosarcina:0.1-1%
  Methanosaeta:0-0%
  Bacteroidales 1:0-0.8%
  Syntrophus:0-0.03%
  Syntrophobacter:0-0.2%
  Sulfurospirillum:0-0.002%
  Methanomethylvorans:0-0.04%
manufacture_protocol: 1 - gDNA was extracted from the mixed culture KB1, 1-3 kb fragments were eluted and purified from 1% agarose gel
  2 - The fragments were then blunt-end repaired and dephosphorylated before undergoing blunt-end cloning.
  3 - After transformation into E. coli, individual bacterial colonies were picked into  plates , and grown over night
  4- PCR amplification of the inserts was performed using the E.Coli culture and T7f/M13r primers for the plasmid
  5 - The PCR products were purified, quantified spectrophotometrically and then transferred to 384-well plate and adjusted to 200 ng/µl in 3XSSC buffer and sent for printing.
  6- The amplicons were spotted with the VersArray ChipWriter Pro System (Bio-Rad, 169-0006) using a 48-pin (SMP-3 pins, ArrayIT, product #SMP3) configuration.
  7 - Each clone was spotted once on GAPS II–coated slides (Corning, 40003).
  8 - The printed arrays were processed following manufacturer’s protocol (Corning) and kept in a dessicator for later use.