SAMN00168373
External Id
LIBEST_025374
External Id
University of British Columbia
INSDC center name
2009-11-17T00:00:00Z
INSDC first public
2023-10-26T21:06:24.433Z
INSDC last update
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
ERS16610514
SRA accession
Grosmannia clavigera (Ophiostoma clavigerum) isolate SLKW1407 (UAMH 11150) was collected in 2003 from a mountain pine beetle gallery in Lodgepole pine (Pinus contorta) near Kamloops, BC. Cultures of this isolate were started from 100 microliters of 10^5 conidia/mL in water on the following media overlaid with cellophane: 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). 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. Mycelium were grown on these plates for six days prior to harvesting. 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 (129 micrograms) was extracted using the Qiagen RNeasy Midi Kit. mRNA (382 nanograms) was purified from the total RNA using the Oligotex mRNA Kit (Qiagen). First strand cDNA was prepared from 115 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 (1 mL) was approx. 4x10^5 cfu/mL and was not amplified.
description
SLKW1407 (UAMH 11150)
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
Ocl03-Kw1407-LppMeOH-Unnorm (OCL03)
title
Plasmid
http://purl.obolibrary.org/obo/SO_0000155
v_type
pDNR-LIB
vector