SAMN00168372
External Id
LIBEST_025373
External Id
University of British Columbia
INSDC center name
2009-11-17T00:00:00Z
INSDC first public
2023-10-26T21:06:24.416Z
INSDC last update
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
ERS16610513
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: Wood (W)- Lodgepole pine sawdust 10g, 1.5percent Granulated Agar; Starch (S)- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Starch, 0.1percent Potassium Hydrogen Phthalate and 0.3percent Asparagine; Organic Nitrogen (ON)- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Maltose, 0.1percent Potassium Hydrogen Phthalate and 0.3percent Asparagine; Inorganic Nitrogen (IN)- 0.17percent YNB, 1.5percent Granulated Agar, 1percent Maltose, 0.1percent Potassium Hydrogen Phthalate and 0.3percent NaNO3; Olive oil (OO)- 0.17percent YNB, 1.5percent Granulated Agar, 1percent (v/v) Olive oil, 0.1percent Potassium Hydrogen Phthalate, 0.5percent tergitol and 0.3percent Asparagine. Mycelium were grown on these plates for six days prior to harvesting. A 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). Total RNA (300 micrograms) was extracted using the Qiagen RNeasy Midi Kit. mRNA (4.7 micrograms) was purified from the total RNA using the Oligotex mRNA Kit (Qiagen). First strand cDNA was prepared from 1 microgram 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). This cDNA was then normalized with duplex-specific nuclease and amplified following the TRIMMER-DIRECT protocol, 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. 2.1x10^5 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
Ocl02-Kw1407-W-S-ON-IN-OO-Norm (OCL02)
title
Plasmid
http://purl.obolibrary.org/obo/SO_0000155
v_type
pDNR-LIB
vector