SAMN00168375
External Id
LIBEST_025376
External Id
University of British Columbia
INSDC center name
2009-11-17T00:00:00Z
INSDC first public
2023-10-26T21:06:24.466Z
INSDC last update
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
ERS16610516
SRA accession
Isolate: ATCC18086, B5 (UAMH 11154), B10 (UAMH 11155), H55 (UAMH 11153), 200_1_14 (UAMH 11151), DPCHMC3 (Breuil culture collection, Dept. of Wood Science, University of British Columbia, Vancouver, Canada), and DPLKGT1B (UAMH 11152). Cultures of Grosmannia clavigera (Ophiostoma clavigerum) isolates ATCC18086, B5 (UAMH 11154), B10 (UAMH 11155), H55 (UAMH 11153), 200_1_14 (UAMH 11151), DPCHMC3 (Breuil culture collection, Dept. of Wood Science, University of British Columbia, Vancouver, Canada), and DPLKGT1B (UAMH 11152) were started from 100 microliters of 10^5 conidia/mL in water on the following media overlaid with cellophane: Wood- Lodgepole pine sawdust 10g, 1.5percent Granulated Agar; Starch- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Starch, 0.1percent Potassium Hydrogen Phthalate and 0.3percent Asparagine; Organic Nitrogen- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Maltose, 0.1percent Potassium Hydrogen Phthalate and 0.3percent Asparagine; Inorganic Nitrogen- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Maltose, 0.1percent Potassium Hydrogen Phthalate and 0.3percent NaNO3; Olive oil- 0.17percent YNB, 1.5percent Granulated Agar, 1percent (v/v) Olive oil, 0.1percent Potassium Hydrogen Phthalate, 0.5percent tergitol and 0.3percent Asparagine; Methanol extract- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Maltose, 0.1percent Potassium Hydrogen Phthalate, 0.3percent Asparagine and 200 microlitres of crude lodgepole methanol pine extract (80:20, MeOH:H2O). Mycelium were grown on these plates for six days prior to harvesting. The crude lodgepole methanol extract was prepared as follows: a lodgepole pine bolt from a freshly cut tree was brought to the lab and then frozen at -20. While still frozen, the bolt was cut into disks and the phloem separated. The phloem was then ground in a mill with liquid nitrogen. The ground phloem was then extracted in 80:20 methanol:water (2.5 mL/g) and sonicated at 4oC for 2 hr. After centrifugation, the supernatant was removed and concentrated by 1/3 under a gentle flow of nitrogen gas. This concentrated crude extract was stored at -20oC for no longer than 8 months prior to use. An un-normalized, full-length enriched, directionally cloned library was prepared with the Creator SMART cDNA Library Construction Kit (Clontech, Mountain View, CA, USA) and the TRIMMER-DIRECT cDNA Normalization Kit (Evrogen, Moscow, Russia). The Evrogen kit was used for some of the protocols and for the CDS-3M primer but this is not a normalized library. Total RNA (357 micrograms) was extracted using the Qiagen RNeasy Midi Kit. mRNA (2.8 micrograms) was purified from the total RNA using the Oligotex mRNA Kit (Qiagen). First strand cDNA was prepared from 700 nanograms of mRNA, SuperScript III reverse transcriptase (Invitrogen), CDS-3M primer (Evrogen), and the SMART IV Oligonucleotide (Clontech). Second strand cDNA was prepared by LD-PCR with Phusion Hot Start DNA Polymerase (Finnzymes, Espoo, Finland), digested with SfiI, size fractionated, and cDNA larger than approx. 500 bp was ligated into SfiI-digested pDNR-LIB (Clontech). The ligation mixture was transformed into ElectroMAX DH10B T1 Phage-Resistant electro-competent cells (Invitrogen). Titre of the primary library (2 mL) was approx. 2x10^6 cfu/mL and was not amplified.
description
mixed
http://www.ebi.ac.uk/efo/EFO_0001271
isolate
E. coli DH10B
lab-host
Leptographium clavigerum
http://purl.obolibrary.org/obo/NCBITaxon_226899
organism
Sfi I
re_1
Sfi I
re_2
Mycelium
http://purl.obolibrary.org/obo/BTO_0001436
tissue-type
Ocl05-multistrain-multimedia-Unnorm (OCL05)
title
Plasmid
http://purl.obolibrary.org/obo/SO_0000155
v_type
pDNR-LIB
vector