I-Ultrasonic Lysis yamasampula we-Drosophila melanogaster

I-Drosophila melanogaster isetshenziswa kakhulu kumalabhorethri njengesibonelo semvelo. Ngakho-ke, kufanele kwenziwe izinyathelo zangaphambi kokuhlaziya ezifana ne-lysis, ukuphazanyiswa kwamaseli, ukukhishwa kwamaprotheni kanye nokugundwa kwe-DNA kwamasampuli e-Drosophila melanogaster. Ama-Ultrasonic dismembrators athembekile futhi ayasebenza futhi angasetshenziswa ukwenza imisebenzi ehlukahlukene efana ne-lysis, isizinda samaprotheni noma ukwahlukaniswa kwe-DNA ngokukhululeka ngokulungisa kuphela imingcele yenqubo ye-ultrasonic. Ama-homogenizers e-Ultrasonic ngaleyo ndlela angamathuluzi aguquguqukayo anebanga elibanzi lokusebenzisa.

I-Ultrasonic Lysis kanye nokukhishwa kwamaprotheni

Drosophila melanogaster is widely used as model organism in biological labs. Find here protocols for lysis, protein extracion and DNA shearing of D. melanogaster samples.I-Lysis, i-solubilization cell, i-homogenization yezicubu kanye nokukhishwa kwamaprotheni yimisebenzi ejwayelekile yokuqhekeka kwe-ultrasonic kumalabhorethri wezinto eziphilayo. Ama-Ultrasonic dismembrators kanye nokuphazamiseka kwamaseli kufanelekile ukuhlanganisa izicubu zezilwane, izinambuzane (isb., IDrosophila melanogaster, C. elegans) noma izinhlobo zezitshalo. Ukusetshenziswa okulandelayo kwe-ultrasonication yi-lysis yamaseli ukumiswa kanye nama-pellets kanye nokukhishwa kwamaprotheni we-intracellular.
I-Ultrasonic lysis kanye nesizinda samaprotheni izinqubo ezinokwethenjelwa kakhulu futhi ezizaliswayo, ezingenziwa ngesisekelo semithetho esetshenzisiwe. Njengoba ukuqina kwenqubo ye-ultrasonic kungashintshwa ncamashi ngamapharamitha we-sonication njenge-amplitude, imodi yokujikeleza / yokushaya kwenhliziyo, izinga lokushisa nesampula yevolumu, uma izivumelwano eziqinisekisiwe zingaphindaphindwa ngomphumela ofanayo kaningi.

Izinzuzo ze-Ultrasonic Sample Preparation

  • ephumelelayo kakhulu
  • Ingashintshwa kusampula ethile yesampula
  • Ifanele noma iyiphi ivolumu
  • Ukwelashwa okungashisi
  • imiphumela ye-reproducible
  • Ilula futhi iphephile

I-Ultrasonic DNA ne-RNA Fragmentation

Ngemuva kokukhishwa kweseli nokukhishwa kwamaprotheni, isinyathelo esivamile esidingekayo ekulungisweni kwesampula ukugunda nokuhlukaniswa kwe-DNA, i-RNA, ne-chromatin, isb ngaphambi kwe-chromatin immunoprecipitation (ChIP). Ukuhlukaniswa kwe-DNA ne-RNA kungafinyelelwa ngokuthembekile ngokwaphula ama-covalent bonds abamba i-DNA ndawonye ngamandla emvelo. Kusetshenziswa ukugunda ngokomzimba njenge-sonication, ekuqaleni imicu ye-DNA iphukile, i-DNA ihlukaniswe yaba izingcezu ezincane.
Ukuhlukaniswa kwe-Ultrasonic DNA kunokwethenjelwa futhi kusebenza kahle ekugundeni i-DNA kubude obuhlosiwe, isb. 500bp (base pair). Izinzuzo ezinkulu zokuhlukaniswa kwe-DNA ye-ultrasonic zifaka ukulawulwa okuqondile kwamapharamitha wenqubo ye-ultrasonic namandla. Imingcele yenqubo ye-ultrasonic ingalungiswa ngokulungisa ukuqina kwe-sonication, imijikelezo nesikhathi ngokunembile. Lokhu kwenza kube nokwenzeka ukudala osayizi be-DNA abafunayo futhi ubude be-DNA obuhlosiwe bungakhiqizwa ngokuthembekile futhi buphinde bukhiqizwe. Ukucheba i-Ultrasonic DNA nakho kuhle ukudala izingcezu eziphezulu ze-DNA zamangqamuzana.

Ultrasonicator UP200Ht with microtip S26d2 for ultrasonic lysis of Drosophila samples

I-Ultrasonicator UP200Ht nge-2mm microtip S26d2 ye-sonication yamasampuli we-Drosophila

Isicelo solwazi




Qaphela kwethu Inqubomgomo yobumfihlo.


Izivumelwano ze-Ultrasonic Lysis zeDrosophila melanogaster

Ngezansi ungathola izivumelwano ezahlukahlukene ze-lysis, ukukhishwa kwamaprotheni, kanye ne-DNA noma ukuhlukaniswa kwe-chromatin kwamasampuli eDrosophila.

I-Ultrasonic Lysis ye-Cross-linking Immunoprecipitation (CLIP) Assay

Isilingo se-CLIP senziwe njengoba kubikwe ngaphambilini ngokunye ukulungiswa. Cishe ama-ovaries angama-20 mg avela kwabesifazane bezinhlobo zasendle ezineminyaka engu-0- kuya ku-1 anezinhlamvu ezixhunywe nge-UV (3 × 2000 μJ / cm2), zahlanganiswa neqhwa ku-1 mL RCB buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.1% Triton X-100, 250 mM sucrose, 1 mM DTT, 1 × EDTA-free Complete Protease Inhibitors, 1 mM PMSF) engezwe nge-300 U RNAseOUT futhi ibekwe eqhweni imizuzu engama-30. I-homogenate yenzelwe iqhwa, ngamandla angu-80%, amahlandla ayisihlanu ngokuqhuma okungama-20 okuphumule okungu-60 phakathi kokusebenzisa iHuelscher Ultrasonic Processor I-UP100H (100 W, 30 kHz) ne-centrifuged (16000 × g ngamaminithi ama-5 ku-4 ° C). Ukukhishwa okuncibilikisiwe kwalungiswa nge-20 μl Amaphrotheni-G dynabeads ngamaminithi angama-20 ku-4 ° C. Ngemuva kokususwa kwamasampuli we-immunoblotting kanye nobungako bokufakwa kwe-RNA (1%), i-HP1 yafakwa amandla omzimba nge-anti-HP1 9A9 antibody kusuka ku-450 μl ekhishwe ngaphambili nge-incubation ye-4 h nge-50 μl Protein-G dynabeads. Ama-immunoprecipitates agezwe amahlandla ama-4 nge-RCB. Ukweqa ama-RNA angenakugwenywa emzimbeni, ubuhlalu obunamaphiko babiliswa ku-100 μL Yamanzi aphethwe yi-UltraPure DEPC amaminithi ama-5. I-900 μL Qiazol Reagent ingezwe kumandla amakhulu atholakele ukulungiselela i-RNA. I-RNA ehlanzwe isetshenziswe njengesifanekiso sokwenza i-cDNA isebenzisa i-oligo dT, ama-hexamers angahleliwe kanye ne-SuperScript reverse transcriptase III ngokuya ngomthetho olandelwayo womenzi.
(UCassale et al. 2019)

I-Ultrasonic Lysis yeChromatin Immunoprecipitation Assay

I-Chromatin immunoprecipitation yenziwa ngokwendlela echazwe yi-Menet enezinguquko ezincane. Cishe ama-ovaries angama-20 mg kusuka kwabesifazane bezinhlobo zasendle ezineminyaka engu-0- kuya ku-1 ubudala ahlanganiswa ngo-1 ml we-NEB buffer (10 mM HEPES-Na ku-pH 8.0, 10 mM NaCl, 0.1 mM EGTA-Na ku-pH 8, 0.5 mM I-EDTA-Na ku-pH 8, 1 mM DTT, 0.5% NP-40, 0.5 mM Spermidine, 0.15 mM Spermine, 1 × EDTA- free Complete Protease Inhibitors) ene-homogenizer / immersers disperser 1 iminithi (ngo-3000 rpm). I-homogenate idluliselwe endaweni yokufaka ingilazi ebisanda kufakwa futhi kwasetshenziswa imivimbo engu-15 ephelele nge-pestle eqinile. Ama-nuclei wamahhala abesefakwa centrifuged ku-6000xg ngemizuzu eyi-10 ku-4 ° C. Ama-pellets aqukethe ama-nuclei aphinda avuselelwa ku-1 mL we-NEB futhi afakwa nge-centrifuged ku-20000 × g ngemizuzu engama-20 ku-sucrose gradient (0.65 mL we-1.6 M sucrose ku-NEB, 0.35 mL we-0.8 M sucrose ku-NEB). I-pellet iphinde yavuselelwa nge-1 mL ye-NEB ne-formaldehyde ekugxileni kokugcina kwe-1%. INuclei yayixhunywe phakathi kwemizuzu eyi-10 ekamelweni lokushisa futhi yacinywa ngokungeza i-1/10 vol ye-1.375 M glycine. Ama-nuclei aqoqwa nge-centrifugation ku-6000 × g ngemizuzu emi-5. INuclei igezwe kabili ku-1 ml we-NEB yaphinde yafakwa ku-1 mL weLysis Buffer (15 mM HEPES-Na ku-pH 7.6, 140 mM NaCl, 0.5 mM EGTA, 1 mM EDTA ku-pH 8, 1% Triton X-100, 0.5 I-mM DTT, i-0.1% i-Na Deoxycholate, i-0.1% i-SDS, i-0.5% ye-N-lauroylsarcosine ne-1 × i-EDTA engenawo ama-Protease Inhibitors angenawo wonke). INuclei yenziwa indodana ngokusebenzisa i-Hielscher Ultrasonic Processor I-UP100H (100 W, 30 kHz) amahlandla ayisithupha kuma-20 s naku-1 min eqhweni. Ama-nuclei e-Sonicated abekwe phakathi kwe-13000 × g ngamaminithi ama-4 ku-4 ° C. Iningi le-chromatin e-sonicated yayingu-500 kuye ku-1000 base pair (bp) ubude. Ku-immunoprecipitation ngayinye, i-15 μg ye-chromatin yafakwa phambi kwe-10 μg ye-HP1 9A9 antibody monoclonal (3 h ku-4 ° C esondweni elijikelezayo). Ngemuva kwalokho, i-50 μl yamaprotheni we-dynabeads G yangezwa futhi ukufukama kwaqhutshwa ubusuku bonke ku-4 ° C. Ama-supernatants alahliwe futhi amasampula ahlanzwa kabili eLysis Buffer (i-wash ngayinye i-15 min ku-4 ° C) futhi kabili ku-TE Buffer (1 mM EDTA, 10 mM TrisHCl ku-pH 8). I-Chromatin yakhishwa ebuhlalu ngezinyathelo ezimbili; okokuqala ku-100 μl ye-Eluition Buffer 1 (10 mM EDTA, 1% SDS, 50 mM TrisHCl ku-pH 8) ku-65 ° C ngemizuzu eyi-15, kulandelwa ukufuthwa ngamandla nokubuyiselwa kwamandla amakhulu. Izinto zobuhlalu ziphinde zakhishwa ngo-100 μl we-TE + 0.67% SDS. I-eluate ehlangene (200 μl) ifukanywe ubusuku obubodwa ngo-65 ° C ukuguqula izixhumanisi ezinqamulelayo futhi iphathwe yi-50 μg / ml RNaseA ngemizuzu engu-15 ngo-65 ° C nange-500 μg / ml Proteinase K ye-3 h ku-65 ° C. Amasampula akhishwa i-phenol-chloroform kwathi i-ethanol yancipha. I-DNA iphinde yavuselelwa ku-25 μl wamanzi. Ukwandisa ukuhlaziywa kwamangqamuzana nge-DNA immunoprecipised, izakhi zofuzo ezikhethiwe zandiswa ngamabili ngoprosesa we-duplex-PCR owenziwe kahle ngokusebenzisa amasethi amabili ahlukene ezinamazinga okushisa ancibilika ngokufana ekuphenduleni okukodwa.
(UCasale et al. 2019)

I-UP200St TD_CupHorn ye-sonication engaqondile yamasampuli

UP200St TD_CupHorn i-sonication engaqondile yamasampuli afana ne-DNA kanye nokugunda kwe-chromatin

Iziphazamisi Zamakhemikhali Ezisebenza Nge-High-Performance zeSampuli Zebhayoloji

I-Hielscher Ultrasonics ingumlingani wakho onolwazi olude uma kukhulunywa ngama-ultrasonicators asebenza kahle kakhulu wokuphazamiseka kwamaseli, i-lysis, ukukhishwa kwamaprotheni, i-DNA, i-RNA, nokuhlukaniswa kwe-chromatin kanye nezinye izinyathelo zokulungiselela isampula zangaphambi kokuhlaziya. Ukunikeza iphothifoliyo ebanzi yama-homogenizers e-ultrasonic lab kanye namayunithi wokulungiselela amasampula, i-Hielscher inedivayisi ekahle ye-ultrasonic yohlelo lwakho lokusebenza nezidingo zakho.
I-probe-type insonifier UP200St ye-lysisI-ultrasonicator yohlobo lwe-classic probe ene-micro-tip efana ne- UP200St (200W; bheka isithombe kwesobunxele) noma enye yeyunithi yokulungiselela isampula ye-ultrasonic I-VialTweeter noma UP200St_TD_CupHorn nge-VialHolder amamodeli ayizintandokazi ocwaningweni nakuma laboratories wokuhlaziya. I-classic ultrasonic probe ilungile, lapho kulungiswa amasampula ambalwa kufanele afakwe ilys, akhishwe noma ahlukaniswe. Amayunithi okulungiselela isampula i-VialTweeter ne-UP200St_TD_CupHorn avumela ukunikezwa kwe-sonication ngesikhathi esisodwa kwezigubhu eziyi-10 noma ezi-5 ngokulandelana.
Uma izinombolo zesampula eziphezulu (isb. Amapuleti angama-96-kahle, amapuleti ama-microtiter njll.) Kufanele kucutshungulwe, I-UIP400MTP ukusetha okuhle kwe-sonication. I-UIP400MTP isebenza njengophondo lwenkomishi olukhudlwana, eligcwele amanzi futhi inesikhala esanele sokubamba amapuleti emithombo emincane. Inikwe amandla yi-400 watts processor enamandla ye-ultrasonic, i-UIP400MTP iletha umfaniswano onamandla kakhulu futhi onamandla wamapuleti anemithombo eminingi ukuze kuphazamise amaseli, amasampula we-lyse, ukuxubungula amaphilisi, ukukhipha amaprotheni noma i-shear DNA.

Ukulawulwa okuqondile ngeSmart Software

Abacwaningi bezezimboni ze-Hielscher ze-hdT uchungechunge bangaba mnandi futhi basebenzise umsebenzisi nge-remote control control.Zonke izixazululo ze-Hielscher sonication ezivela kuma-watts angama-200 kuya phezulu zine-digital color touch-screen kanye nesoftware ehlakaniphile. Nge-smart data protocolling yonke imingcele yenqubo ye-ultrasonic igcinwa ngokuzenzekelayo njengefayela le-CSV ekhadini le-SD elakhelwe ngaphakathi ngokushesha nje lapho i-ultrasonicator iqalwa. Lokhu kwenza ucwaningo kanye neprotocol kube lula kakhulu. Ngemuva kokuhlolwa kwe-sonication noma ukulungiselela isampula, ungamane ubuyekeze imingcele ye-sonication ye-sonication run ngayinye bese uyiqhathanisa.
Ngokusebenzisa imenyu enembile, ipharamitha eminingi ingasethwa kabusha ngaphambi kwe-sonication: Isibonelo, ukulawula izinga lokushisa kusampula nokuvimbela ukonakala kwalo okushisayo, kungasethwa umkhawulo ophezulu wokushisa kwesampula. Inzwa yokushisa engaxhuma, eza neyunithi ye-ultrasonic, inika impendulo ye-processor ye-ultrasonic ekushiseni kwangempela kwe-sonication. Lapho umkhawulo wokushisa ophezulu ufinyelelwa, idivayisi ye-ultrasonic iyama kuze kube yilapho umkhawulo ophansi we-set ∆T ufinyelelwa bese uqala bese u-sonicating ngokuzenzekelayo futhi.
Uma i-sonication ene-input energy ethile iyadingeka, ungasetha kabusha amandla wokugcina we-ultrasonic we-sonication run. Vele, i-ultrasonic pulsation nemodi yokujikeleza nayo ingasethwa ngawodwana, nayo.
Ukuze uphinde usebenzise imingcele ye-sonication ephumelele kakhulu, ungagcina izindlela ezahlukahlukene ze-sonication (isb. Isikhathi sonication, umfutho, imodi yokujikeleza njll.) Njengezindlela zangaphambi kokusetha, ukuze kube lula futhi ziqale ngokushesha futhi.
Ukuze kube lula ukusebenza, wonke amayunithi edijithali e-ultrasonic angaqhutshwa nge-browser elawulwa kude kunoma yisiphi isiphequluli esivamile (isb., InternetExplorer, Safari, Chrome njll). Ukuxhumeka kwe-LAN ukusetha okulula kwe-plug-n-play futhi akudingi ukufakwa kwesoftware okwengeziwe.
Thina kwaHielscher siyazi ukuthi ukwenziwa ngempumelelo kwamasampuli wezinto eziphilayo kudinga ukunemba nokuphindeka. Ngakho-ke, senzelwe ama-ultrasonicators ethu njengamadivayisi ahlakaniphile anazo zonke izici ezenza ukulungiselela ukulungiselela okusebenzayo, okunokwethenjelwa, okuzalayo futhi okulula.

Xhumana nathi manje futhi usitshele ngamasampuli akho ezinto eziphilayo kanye nezinyathelo ezidingekayo zokuzilungiselela. Sizokuphakamisa ukuthi ulungiselele isampula yedivayisi elungele kakhulu futhi sikusize ngolwazi olungeziwe olufana nezivumelwano nezincomo.

Ithebula elingezansi likunikeza inkomba yamandla wokucutshungulwa kwesistimu yethu ye-ultrasonic kusuka kuma-ultrasonic micro-tips kanye ne-classic homogenizers ye-ultrasonic kuya kuma-MultiSample ultrasonicators ukulungiselela okulula, okuthembekile kwamasampuli amaningi:

Iqoqwana Ivolumu flow Rate Amadivayisi ezinconyiwe
Amapuleti angama-96-kahle / ama-microtiter na I-UIP400MTP
Izitsha eziyi-10 kuya ku-0.5 kuye ku-1.5mL na I-VialTweeter ku-UP200St
I-CupHorn ye-sonication engaqondile, isib na UP200St_TD_CupHorn
0.01 kuya ku-250mL 5 kuye ku-100mL / min UP50H
0.01 kuya ku-500mL 10 kuya ku-200mL / min UP100H
0.02 kuye ku-1L 20 kuya ku-400mL / min UP200Ht / UP200St
10 kuya ku-2000mL 20 kuya ku-400mL / min UP200Ht, UP400St
0.25 kuya ku-5L 0.05 kuye ku-1L / min UIP500hdT

Xhumana nathi! / Cela Us!

Cela ulwazi oluthe xaxa

Uyacelwa ukuthi usebenzise ifomu elingezansi ukucela ulwazi olungeziwe mayelana nezinhlelo zokusebenza ze-ultrasonic, izinhlelo zokusebenza nentengo. Sizokujabulela ukuxoxa ngenqubo yakho nawe futhi sikunikeze uhlelo lwe-ultrasonic oluhlangabezana nezidingo zakho!









Sicela uqaphele wethu Inqubomgomo yobumfihlo.


The VialTweeter is a MultiSample Ultraonicator that allows for reliable sample preparation under precisely controlled temperature conditions.

Iyunithi yokulungiselela amasampula amaningi we-ultrasonic I-VialTweeter ivumela ukunikezwa kwe-sonication ngasikhathi sinye kwezigubhu eziyi-10. Nge-clamp-on device VialPress, amashubhu angeziwe angama-4 angacindezelwa ngaphambili ukuze kube ne-sonication enamandla.

Izincwadi / Izikhombo



Hielscher Ultrasonics supplies high-performance ultrasonic homogenizers from lab to industrial size.

Ukusebenza okuphezulu kwe-ultrasonics! Uhla lomkhiqizo kaHielscher lumboza isibonisi esigcwele kusuka ku-compact lab ultrasonicator ngaphezulu kwamayunithi ebhentshini-phezulu kuya ezinhlelweni ezigcwele zezimboni ze-ultrasonic.


Amaqiniso Okufanele Ukwazi

Metabolomics

I-Metabolomics ukucwaninga kwama-molecule amancane, aziwa ngokuthi ama-metabolites, akhona ngaphakathi kwamaseli, ama-biofluids, izicubu noma izinto eziphilayo. Lawa ma-molecule amancane kanye nokusebenzisana kwawo ohlelweni lwezinto eziphilayo kufingqiwe ngaphansi kwegama lesambulela elithi "metabolome" futhi umkhakha wocwaningo ubizwa ngokuthi yi-metabolomics. Ucwaningo lwe-metabolome luxhumene kakhulu nomkhakha oqhamuka ngokushesha womuthi wokunemba. Ukuqondwa kwemetabolome kanye nokuhlobana kwayo nezifo ezahlukahlukene kusiza ukuthuthukisa izindlela zokuvimbela izifo kanye nezindlela zokunakekelwa kwemitholampilo ngenkathi ukuhlukahluka komuntu ngamunye kwezemvelo, indlela yokuphila, izakhi zofuzo, kanye ne-phenotype yamangqamuzana. Ukuze kukhululwe ama-molecule e-metabolite kusuka kumaseli, i-ultrasonication isetshenziswa njalo kuma laboratories ebhayoloji ukulungiselela amasampula angaphambi kokuhlaziya njengokuphazanyiswa kwamaseli, i-lysis kanye nokukhishwa kwamaprotheni, i-lipids namanye ama-molecule.