@prefix pplan: <http://purl.org/net/p-plan#> .
@prefix this: <http://purl.org/np/RA3MmCHHYDKYpNg2eP3Tmjn2QR_wpZXHhDjJnnCsuLFY8> .
@prefix sub: <http://purl.org/np/RA3MmCHHYDKYpNg2eP3Tmjn2QR_wpZXHhDjJnnCsuLFY8#> .
@prefix bs: <https://bioschemas.org/profiles/LabProtocol/0.4-DRAFT-2020_07_23/#> .
@prefix np: <http://www.nanopub.org/nschema#> .
@prefix pav: <http://purl.org/pav/> .
@prefix authority: <https://hypothes.is/a/> .
@prefix npx: <http://purl.org/nanopub/x/> .
@prefix xsd: <http://www.w3.org/2001/XMLSchema#> .
@prefix rdfs: <http://www.w3.org/2000/01/rdf-schema#> .
@prefix prov: <http://www.w3.org/ns/prov#> .
@prefix sp: <http://purl.org/net/SMARTprotocol#> .
@prefix dc: <http://purl.org/dc/elements/1.1/> .
sub:Head {
   this: np:hasAssertion sub:assertion ;
    np:hasProvenance sub:provenance ;
    np:hasPublicationInfo sub:pubInfo ;
    a np:Nanopublication .
}
sub:assertion {
   <http://purl.obolibrary.org/obo/CHEBI_17992> rdfs:label "100mM sucrose" .
  <http://purl.obolibrary.org/obo/CHEBI_29234> rdfs:label "1% Triton -X" .
  <http://purl.obolibrary.org/obo/CHEBI_8984> rdfs:label "1% SDS" .
  sub:_1 rdfs:label "mosquitoes" .
  sub:_2 rdfs:label "mosquitoes" .
  sub:_3 rdfs:label "micropistle" .
  sub:_4 rdfs:label "Rnase A (20mg/ml)" .
  sub:_5 rdfs:label "1X STE buffer" .
  sub:_6 rdfs:label "Proteinase K (20mg/ml)" .
  sub:step dc:description "Grind mosquitoes  in  1.5 ml eppendorf  tube with micropistle in 50-100 µl 1X STE buffer ( 50mM Nacl,50mM Tris- HCL,100mM EDTA, Ph 8.0) along with 100mM sucrose. Add 1X STE buffer to a total volume of 300-500 µl for a single mosquito and 1 ml for mosquito pool like 4,6,8,10 numbers. Then add 1% SDS ,1% Triton -X , 10 µl/ ml  Rnase A (20mg/ml), 20 µl/ ml  Proteinase K (20mg/ml) and mix it." ;
    sp:hasExperimentalInput sub:_1 ;
    a pplan:Step ;
    bs:bioSampleUsed sub:_2 ;
    bs:labEquipmentUsed sub:_3 , <http://www.bioassayontology.org/bao#BAO_0010014> ;
    bs:reagentUsed <http://purl.obolibrary.org/obo/CHEBI_17992> , <http://purl.obolibrary.org/obo/CHEBI_29234> , <http://purl.obolibrary.org/obo/CHEBI_8984> , sub:_4 , sub:_5 , sub:_6 .
  <http://www.bioassayontology.org/bao#BAO_0010014> rdfs:label "1.5 ml eppendorf  tube" .
}
sub:provenance {
   sub:assertion pav:createdWith <https://nanotate.bitsfetch.com> ;
    prov:generatedAtTime "2021-03-16T16:48:04.108013"^^xsd:dateTime ;
    prov:wasAttributedTo <https://hypothes.is/users/iannotate> .
}
sub:pubInfo {
   sub:assertion prov:wasDerivedFrom authority:8Y0HtIJ9EeuxMKftfvVUrw , authority:AHbk6IJ-EeuLn7c9eitXuQ , authority:CWoxtIahEeueMafFr2xIRQ , authority:FBJa-oJ-EeuVON8E0bPEnQ , authority:GGaOiIahEeuEzbud0kuutg , authority:JzPoLoJ-EeubeTMBRz-oHg , authority:P9yYHIJ-Eeuk1J8X-v5LOg , authority:VhkMtIJ-EeuaQsuiGJkaPw , authority:dCvMjIJ-EeuYqLMMrOutfQ , authority:i-hYaIJ-EeuaRdNsg6w5Hg .
  sub:sig npx:hasAlgorithm "RSA" ;
    npx:hasPublicKey "MIGfMA0GCSqGSIb3DQEBAQUAA4GNADCBiQKBgQCEOtYac15zsWN/LAu3q7YFdW0UZNfX3BAarHW/LB1DHGcz0yYr0v7hKGbnCLJ+UkDfu3Mgj7BnCm1s8ulMULKfF4YjqS2YKHYZjNKVaIjSigFOpjOwIx99+qPlvd5CuG+Gia/sJA05jy2bPKLwb/kmVeeX0zORZudWLyRMLXp8UQIDAQAB" ;
    npx:hasSignature "aZd/AL+wAll/02gNVAmhv8QGPBCTs7otfD4kaMz6CzndlDewoaVZcCmCBsl0xltZZZisvKm9bu+mhYyIwYX+5z4L3rxcyLp7AxPMD7roKQ7W9/5gRzY5DSAJH9kE9Y6jZKoTkDGwyXty1qOddP/83ut8S3q0w1HrYrcGVrpP6OU=" ;
    npx:hasSignatureTarget this: .
  this: npx:introduces sub:step ;
    prov:generatedAtTime "2021-03-16T16:48:04.108013"^^xsd:dateTime ;
    prov:wasAttributedTo <https://hypothes.is/users/iannotate> .
}