I-Hielscher Ultrasound Technology

Ukukhishwa kwe-Ultrasonic DNA

  • Phakathi ne-DNA ne-RNA ukugqoka, ama-molecule e-DNA aphukile abe izingcezu ezincane. Ukuhlukaniswa kwe-DNA / RNA kungenye yesampula ebalulekile isampula prep izinyathelo zokudinga ezidingekayo zokudala ukulandelana kwesizukulwane esilandelayo (NGS).
  • I-ultrasonic DNA ukugqoka isebenzisa amandla e-acavtic cavitation ukuphula i-DNA noma i-RNA zibe izingcezu eziyikhulu – 5kb bp.
  • Ukukhipha i-ultrasonic kuvumela ukuhlukaniswa kwe-DNA eqondile nokuguqula i-DNA ubude obude.

Ukukhishwa kwe-Ultrasonic DNA

I-Hielscher Ultrasonics inikeza izixazululo ezihlukahlukene ezisekelwe nge-ultrasound ze-DNA, i-RNA ne-sherom chromatin. Khetha phakathi kwe-proxy-type ultrasonicators (isib. UP100H) ye-sonication ngokuqondile usebenzisa i-microtip, noma usebenzise i-VialTweeeter noma i-cuphorn ye-ultrasonic ye-DNA yokulungiselela amashiyi ahlukahlukene ngesikhathi esisodwa. I-Hielscher inikeza idivaysi ekahle ngokucabangela izidingo zakho: unamanzi unama-sampuli angu-1 noma angama-10, amanani avela ku-microliter kuya ku-litre – Amaphrosesa ase-Hielscher ultrasonic ayatholakala ukuze ahlangabezane nezidingo zakho ukuze ulungiselele ama-DNA, i-RNA nama-chromatin fragments ngesikhatsi esifanele. I-Reproducibility, ukusebenza okulula nokulawula okuqondile kuvumela umtapo onokwethenjelwa wokulandelana kwesizukulwane esilandelayo.
Ngokuphambene nokuhlukaniswa kwe-DNA enzymatic, ukugcoba kwe-ultrasonic kusebenza okuhlanzekile kwe-shear force ngaphandle kokungeza noma yimaphi amakhemikhali. Ngokusekwa ngokucacile kwezinqubo zemigomo, ukugcoba kwe-ultrasonic kuveza izingxenyana eziphakeme ze-DNA ze-DNA (i-plasmid ne-genomic DNA).
I-nucleic acids ehlanjululwe ingahle yenziwe ngaphambi noma ngemuva kwesinyathelo sokuhlukaniswa.
Imingcele ye-Sonication (amandla, umjikelezo we-pulse / bursts, isikhathi nendawo yokushisa) ingalawulwa ngokuphepha ngezilungiselelo zesofthiwe.

Izinzuzo:

  • ukulawula okuqondile
  • imijikelezo ye-Sonication nesikhathi esivumelana kahle nesayizi efunwa yi-DNA
  • ama-molecular isisindo somzimba we-DNA
  • ukushisa lokushisa
  • Okusheshayo
  • imiphumela ye-reproducible
  • i-autoclavable
  • izixazululo ezihlukahlukene: Uhlobo lwe-Probe, I-VialTweeter futhi CupHorn

Izivumelwano ze-Ultrasonic DNA Shearing

I-Chromatin Immunoprecipitation Assay

Ngamafuphi, amangqamuzana ahlanganiswe izitsha ezingu-60mm-diameter (400,000 per dish) futhi ahanjiswe nge-RhoA siRNA (njengoba kuchaziwe); emva kuka-72 h, babekwe nge-formaldehyde (ukuhlushwa kokugcina, 1%) ngamaminithi angu-10 ku-37 ° C ukuze kutholakale amaprotheni angenelela ku-DNA. Ukusabela okuxhumanisa isiphambano kwaqedwa ngokungezwa kwevolumu eyodwa-yesisindo se-1.25 mol / L glycine, enikeza ukugxila kokugcina kuka-125 mmol / L. Amaseli ahlanzwa kabili nge-PBS ebandayo eqhwa, aphinda aphinde abuyele esimweni se-radioimmunoprecipitation assay buffer [150 mmol / L NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 5 mmol / L EDTA, 50 mmol / L Tris-HCl (pH 8.0 )] equkethe 1 mmol / L phenylmethylsulfonyl fluoride, 1 Ag / mL aprotinin, ne-1 Ag / mL pepstatin A, futhi igcinwe ngeqhwa ngamaminithi angu-30. Khona-ke, amangqamuzana e-cell ahanjiswa ngeqhwa nge-a Hielscher UP200S i-ultrasound sonicator (3 x 40 s, i-amplitude 40%, umjikelezo 1; i-Hielscher Ultrasonics GmbH) kuze kufike ama-chromatini axhunyiwe ahambise izidakamizwa ze-DNA phakathi kuka-200 no-1,000 bp. Ingxenye yeshumi ye-lysate yonke yayisetshenziselwa ukulinganisa inani le-DNA elitholakala kuma-sampuli ahlukene futhi kubhekwa njenge “isamba se-DNA yokufaka”. Ama-supernatants ayenziwe nge-salmon sperm DNA / amaprotheni agarose-50% slurry ukunciphisa isizinda esingenasici. I-immunoprecipitation yenziwa njalo ebusuku ngo-4 ° C nge-5 Ag ye-anti-NF-nB p65 (i-Upstate) noma ngaphandle kwe-antibody (ukulawulwa okubi). Lezi zinsikazi zazixhaswe nge-5 mol / L NaCl futhi zivutha ubusuku obuneminyaka engama-65 ° C ukuze zibuyisele izixhumanisi ezihamba phambili ze-protein-DNA. Ama-immunocomplex aphinde aphathwe nge-DNase- no-RNase-free proteinase K, ne-DNA yahlanzwa yi-phenol / chloroform isizinda kanye ne-ethanol precipitation. I-PCR yenziwe ngezinhlamvu ezithile ezihambisana nokulandelana ngaphakathi kwesifunda sokugqugquzela isakhi sofuzo se-iNOS (i-p1 primer: 5¶-GAGGGCTTTCCCA- GAACCAAG-3¶; i-p2 primer: GCTGGGCTACTGACCCAG- CAGTTCCAG-3¶). (Doublier et al., 2008)

Ucwaningo lwe-EGFP

Ukuhlola okukhulunywe ngakho, uhlobo oluthile olubuyiselwayo L. tarentolae p10 :: F9Begfp1.4dBsat # 12 (Jena Bioscience, eJalimane) ngesiguli se-EGFP (i-Progressive Green Fluorescent Protein), i-chromosomal ssu ehlangene, yahlonywa emithonjeni ehlukahlukene njengoba kuchazwe ngaphambilini ngaphezu kwalokho kufakwa nge-100 mg l-1 I-Nourseothricin (iJena Bioscience, eJalimane). Ngesikhathi sokutshalwa, amasampuli ama-1 ml athathwe, axhunyiwe ngamakhulu (2000 × g, 20 ° C, 10 min) futhi wageza nge-0.9% isisombululo se-NaCl. I-pellet yaphinda iphinde isetshenziswe (i-HMM 20mM, i-5 mM EDTA, i-2 mM DTT) futhi ihlukaniswe ngokufaka indodana ngeprosesa ye-ultrasonic UP400S (ukusetshenziswa kwamandla ~ 400 Ws). Ama-debris e-cell asuswe yi-centrifugation (6000 × g, 4 ° C, 5 min) futhi ahlaziywe yi-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (i-SDS-PAGE) ngaphansi kokunciphisa izimo ngendlela yeLaemmli (1970) ene-12.5% ​​i-polyacralamide gels . I-EGFP-inkulumo ihlolwe ngesiko elihlukumezekile. (Fritsche et al. 2007)

Hielscher's UP100H was used to prepare EHEC DNA for chip array analysis

Ukuhlaziywa kwe-Electrophoretic ye-DNA ye-genomic ka-E. coli EDL933 inamaminithi angu-0 kuya kwangu-15 ama-ultrasonication. L ikhombisa i-DNA Ladder. (Basselet et al. 2008)

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Qaphela kwethu Inqubomgomo yobumfihlo.


I-Chromatin immunoprecipitation

I-ultrasonic cell iphazamisa u-UP100H (100W) for lysis, ukuphazanyiswa kwamaseli kanye nokugqoka i-DNA.I-chromatin immunoprecipitation test yenziwa nge-ChIP-ITTM I-Express (i-Active Motif, i-Carlsbad, i-CA, eU.SA) ngokusho kwemiyalo yomkhiqizi kanye nokunye ukuguqulwa. Ngamafuphi, ama-podocyte abantu ahlukaniswe ahlanganiswe ne-1% e-formaldehyde ngamaminithi angu-10 ekamelweni lokushisa. Amaseli ahlanzwa nge-PBS ebandayo futhi ukusabela kokulungiswa kwamiswa ngokufaka u-0.125 M glycine wamaminithi angu-5 ekamelweni lokushisa. Amaseli ahlanjululwa futhi nge-PBS ebandayo eqhwa futhi ehlushwa esitsheni. Amaseli aphethwe yi-centrifugation futhi abuye aphinde abuyele esikhungweni se-lysis. Ngemuva kwe-centrifugation, i-nuclei enezintambo zaphinda ziphinde zakhiwe esikhwameni sokugcoba, zifakwe eqhweni ngamaminithi angu-30 futhi i-chromatin yenziwe nge-sonication, isb. UP100H (I-Hielscher Ultrasonics GmbH, iTeltow, eJalimane) ngamandla angu-25% ama-pulses ama-20 sec ngayinye kwiqhwa zibe yizingxenye ezingaba ngu-200-600 bp. I-chromatin ye-sheared yayiyi-centrifuged futhi i-supernatant yaqoqwa. Ukuze i-immunoprecipitations, i-chromatin engu-60 yayihlanganiswe ne-1 μg ye-Sp1 (i-Santa Cruz Biotechnology, i-Santa Cruz, i-CA, i-USA), i-NF-κB p65 (i-Abcam, i-Cambridge, i-UK) noma i-NF-κB p50 (ama-Abcam) IgG (Zymed Laboratories, eSan Francisco, CA, eU.SA), njengendlela yokulawula okungalungile, ubusuku bonke ngo-4 ° C ngokushintshanisa kahle. Ama-immunocomplex ahlanganiswe nobuhlalu bemagnetic aqoqwe esebenzisa i-magnetic stand, ahlanziwe kakhulu, futhi amaprotheni / i-DNA crosslinks aguquliwe futhi i-DNA ihlaziywa ngokuhlaziywa kwe-PCR yangempela. (Ristola et al. 2009)

Ukulungiselela i-EHEC DNA ye-chip array analysis

Ukuhlelwa kwe-lysates yamaseli kanye nama-DNA asusiwe
Amapulangwe angama-bacterium amiswe ePBS kuya ekuhlungeni okugcina okufunayo ayephathwa ngawo i-ultrasound iphazamisa u-UP100H (Hielscher GmbH, eJalimane) enezinsizakalo ze-MS1 (1mm ububanzi). Imvamisa yokusebenza yayingu-30 kHz futhi amandla okukhishwa okuphumelelayo ayengu-100 W. Ngesikhathi sokusebenza, amasampuli ayehlile ebhodini lokugezela ngamanzi e-ice, ahlanganiswe futhi akhululekile. Amasampuli asetshenziselwa ukuqhutshwa kwe-cytometry izifundo, ngenkathi ekuphatheni kwesikhashana, amasampuli aphethwe ukwelashwa okushisa (95 ° C, 5 min). Ama-cell lysates angcolile ahlanganiswa ngenhlanganisela ye-phenol: i-chloroform: isoamyl utshwala (25: 24: 1). Umthamo olinganayo walo mxube wanezelwa kwisampula lysate, isixazululo sasivinjiswe ngamandla amakhulu angu-15 sino-centrifuged at 15,000 xg for 2 min ekamelweni lokushisa (RT) cishe nge-22 ° C. Isigaba esiphezulu se-aqueous equkethe i-DNA ye-genomic sasihlukanisiwe ngokucophelela futhi siqoqiwe emgqonyeni omusha we-Eppendorf oyinyumba.
Kamuva, amasampuli ahanjiswa ngechungechunge leDNA. Isinyathelo sonication safezeka ezimweni ezifanayo njengoba kuchazwe ngenhla. Ukuhlola imiphumela yokuhlukaniswa kwi-DNA ye-genomic, amasampuli ahlaziywa ngokusebenzisa i-agarose gel electrophoresis.
(…) Ama-sampuli azinikezwe ngaphambilini ngamaminithi angu-2.5 ayebhekene nesinyathelo sokukhishwa ngemuva kokunakekelwa kokushisa kanye ne-centrifugation. I-DNA yakhululwa yenziwa izikhathi ezimbili nge-phenol: i-chloroform: i-isoamyl mix mix, futhi kamuva ilandelwa ukunikezwa kwe-secondication kwe-0 - 15 min. I-Agarose gel electrophoresis isetshenziselwa ukunquma ukusabalalisa usayizi we-DNA ebizwa ngokuthi ukuhlukaniswa kwe-ultrasonic ngemuva kokukhipha (isib. Ngakwesokudla phezulu). I-DNA ehlukanisiwe kakhulu ibonakala ngokutholakala kwe-DNA smear kunezingxenyana eziphakeme zamangqamuzana eziqedwe ngamasampuli azinikezelwe ngamaminithi angu-2.5 noma ngaphezulu. I-sonication ende iyancipha kancane kancane ubude bezingcezu kuya ku-150 kuya ku-600 bp, futhi ukunikezwa nge-min minithi okungaphezulu kwezingu-15 kubuye kuphuculise lezi zingcezu, njengoba kungabonakala kakhulu engxenyeni engenhla ye-smear. Ngakho-ke, usayizi wesilinganiso se-DNA we-fragment kancane wehla ngenkathi ye-ultrasonication futhi ukwelashwa okungamaminithi angu-5 kuvunyelwe ukuthola ubukhulu bezingxenye ze-DNA ezifanele kakhulu ezilinganisweni ze-chip array. Ekugcineni, inqubo yokulungiselela ukuhlaziywa kwe-DNA ehlanganisa ukwelashwa kwe-ultrasonic yokuqala kokuqala, i-DNA extraction (2 ×), kanye no-5 minutes sonication, yasungulwa. (Basselet et al. 2008)

I-Chromatin Immunoprecipitation (i-ChIP)

Iprosesa ye-ultrasonic UP100H ye-DNA, i-RNA ne-sherom chromatin. (Chofoza ukuze ukhulise!)Ama-HEK293 amangqamuzana ahlonywe njengoba achazwe ngenhla futhi ahleliwe no-2mM disuccinimidyl-glutarate ngamaminithi angu-45 ekamelweni lokushisa. Kamuva, amaseli ahlanzwa kabili nge-PBS. I-Chromatin yayixhunyaniswe ngemaminithi angu-10 ekamelweni lokushisa besebenzisa i-1% (v / v) i-formaldehyde futhi ihlanzwe kabili nge-PBS ebandayo. Ukusabela okuxhumanisa ukuvinjelwa kwamiswa kwafakwa ngokufakelwa ngamafutha nge-glycine ekugxileni kokugcina kuka-0.125 M nge-5 min ekamelweni lokushisa. Ngemuva kokugxilwa nge-trypsin, amangqamuzana adutshulwa esiteshini samasiko esitokisi futhi ahlanza kabili nge-PBS. I-pellet yeseli yaphinde yabuyekezwa ku-lysis buffer (amapayipi ama-5mM, i-pH 8.0, i-KCl engu-85mM, no-0.5% (v / v) i-Nonidet P-40), ihlanganiswa eqhweni ngamaminithi angu-10, futhi ihambisana ne-homogenizer ye-Dounce. Kamuva, ama-nuclei ayethwetshwe yi-centrifugation (3500 xg, 5 min, 4 ° C) futhi abuye aphinde abuyele e-nuclei buffer (50mM Tris-HCl, i-pH 8.1, i-10 mM EDTA, ne-1% (w / v) i-SDS). I-Nuclei yaphazanyiswa yi-sonication enezintambo ezingu-20-s ku-a I-sonicator ye-UP50H (I-Hielscher Ultraschall Technologie) esimweni somjikelezo we-0.5 nangama-amplitude angu-30%, enikela iziqephu ze-DNA zomsindo ngobukhulu obukhulu be-200 - 1000 bp. Ukuze i-ChIP, i-50g ye-DNA yahlanjululwa ngokuphindwe kabili ekukhunjweni kwe-immunoprecipitation (16.7mM Tris-HCl, i-pH 8.1, 167mM NaCl, i-1.2mM EDTA, i-1.1% (v / v) i-Triton X-100, ne-0.01% (w / v) i-SDS). (Weiske et al. 2006)

Ukuhlaziywa kwe-Histone ukuguqulwa kwe-chromatin immunoprecipitation (i-ChIP)

Ngamafuphi, 6 x 106 amangqamuzana ahlanjwe kabili nge-PBS futhi ahlanganiswa epulatifeni yesikhungo ngamaminithi angu-15 ekamelweni lokushisa phambi kuka-0.5% e-formaldehyde. Ukuphendula okuphambene nomgwaqo kwamiswa ngokungeza u-0.125 M glycine. Zonke izinyathelo ezilandelayo zenziwa ngo-48 ° C. Wonke ama-buffers ayengaphambi kokugcoba futhi aqukethe i-protease inhibitors (Complete Mini, i-Roche). Amaseli ahlanjwe kabili nge-PBS bese ekhishwa. Ama-pellets aqoqiwe ahlakazwa ku-1 ml i-lysis buffer (1% i-SDS, i-5 mM EDTA, i-50 mM i-Tris pH 8) futhi yahanjiswa ebhodini elibandayo le-ethanol imijikelezo engu-10 ku-100% amplitude esebenzisa I-sonicator ye-UP50H (Hielscher, Teltow, eJalimane). Ukuhlukaniswa kwe-Chromatin kuboniswe ku-1% i-gel agarose. Izindwangu ezitholiwe ziphakathi kobubanzi obungama-200-500pb. I-chromatin ene-soluble itholwe ngokufaka i-centrifuging amasampula angenayo ku-14,000g ngamaminithi angu-10 ku-48 ° C. Ingxenyana encibilikile yahlanjululwa i-1/10 ku-dilution buffer (1% i-Triton X-100, i-2 mM EDTA, i-20mM i-Tris pH 8, i-150 mM i-NaCl) bese i-aliquoted futhi igcinwe ku-80 ° C kuze kusetshenziswe. (Rodriguez et al. 2008)

Idivayisi Amandla [W] Thayipha Ivolumu [mL]
I-VialTweeter 200 ukuma 0.5 1.5
UP50H 50 ifakwe ngesandla noma imisiwe 0.01 250
UP100H 100 ifakwe ngesandla noma imisiwe 0.01 500
UP200Ht 200 ifakwe ngesandla noma imisiwe 0.1 1000
UP200St 200 imisiwe 0.1 1000
UP400St 400 imisiwe 5.0 2000
CupHorn 200 I-CupHorn, i-sonoreactor 10 200
I-GDmini2 200 ukungcoliswa-free flow cell

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Qaphela kwethu Inqubomgomo yobumfihlo.


Hielscher's VialTweeter is is´deal for the simultaneous preparation of multiple samples

I-VialTweeter for ultrasonic isampula prep

Xhumana nathi! / Cela Us!

Cela ulwazi oluthe xaxa

Sicela usebenzise ifomu elingezansi, uma ufisa ukucela ulwazi oluthe xaxa mayelana ne-homogenization yama-ultrasonic. Sizojabula ukukunikeza uhlelo lwe-ultrasonic ukuhlangabezana nezidingo zakho.









Sicela uqaphele wethu Inqubomgomo yobumfihlo.


Izincwadi / Izinkomba

  • I-Basselet P., Wegrzyn G., i-Enfors S.-O., iGabig-Ciminska M. (2008): Ukucubungula kwesampula se-DNA kuhlaziywa ngokusekelwe kwe-Escherichia coli (EHEC). Ama-Microbial Cell Factories 7:29. 2008.
  • Doublier S., Riganti Ch., Voena C., Costamagna C., Aldieri E., Pescarmona G., Ghigo D., Bosia A. (008): I-RhoA Silencing Ibuyisela Ukuphikiswa Kwe-Doxorubicin Ezingqamuzaneni Zomdlavuza Wamuntu. Ucwaningo lwe-Cancer Molecular 6 (10), 2008.
  • U-Fredlund E., uGidlund A., u-Olsen M., uBörjesson T., u-Spliid NHH, uSimonsson M. (2008): Ukuhlolwa kokusebenza kwe-Fusarium DNA ukukhipha kusuka ku-mycelia nokukolweni ukwenzela ukuthi kutholakale isikhathi se-PCR esilinganiselwe kanye nokulungiswa kwamanothi e-mycotoxin . I-Journal of Microbiological Methods 2008.
  • UFritsche C., uSitz M., Weiland N., uBreitling R., uPohl H.-D. (2007): Ukufaniswa kokuziphatha kokukhula kweLeishmania tarentolae - uhlelo olusha lwezinhlelo zamaphrotheni avuselelayo. Journal of Basic Microbiology 47, 2007. 384-393.
  • Ristola M., Arpiainen S., Saleem MA, Mathieson PW, Welsh GI, Lehtonen S., Holthöfer H. (2009): Ukulawulwa kwe-Neph3 geni kuma-podocytes - izindima ezibalulekile zezinto ezibhaliwe NF-κB kanye ne-Sp1. BMC Biology Biology 10:83, 2009.
  • U-Rodriguez J., Vives L., Jorda M., Morales C., Munoz M., Vendrell E., Peinado MA (2008): Ukulandela ngokulandelana kwe-DNA i-DNA Alu engaphenduliwe emaselini avamile kanye nomdlavuza. I-Nucleic Acids Research Vol. 36, No. 3, 2008. 770-784.
  • I-Weiske J. Huber O. (2006): I-Histidine Triad Protein I-Hint1 ivuselela i-Apoptosis i-Independent Yomsebenzi wayo we-Enzymatic. I Journal of Biological chemistry. I-Vol. 281, No. 37, 2006. 27356-27366.


Amaqiniso Okufanele Ukwazi

I-Ultrasonic / acoustic cavitation idala amandla amakhulu kakhulu akhuthaza izinqubo ze-crystallization kanye nezulu (Chofoza ukuze ukhulise!)

I-ultrasonic DNA ukugqoka isekelwe cavitation acoustic kanye namandla yayo hydrodynamic shear