EndoFree Plasmid Maxi Kit

Yanayin ajiya

Ya kamata a adana RNaseA a -30 ~ -15 ℃ kuma a kai shi a ≤0 ℃.

Ana iya adana Scavenger na Endotoxin a 2 ~ 8 ℃ na wata daya, adana a -30 ~ -15 ℃ don adana dogon lokaci.kuma ana jigilar su a ≤0℃.

Sauran abubuwan da aka gyara yakamata a adana su a dakin da zafin jiki (15 ~ 25 ℃) kuma a kai su cikin zafin jiki.

Abubuwan da aka gyara

RNaseA:10 mg/ml, ana amfani dashi don cire RNA.

Buffer P1:buffer dakatarwar kwayan cuta, ƙara RNaseA zuwa Buffer P1 kafin amfani da farko.

Buffer P2:buffer lysis na kwayan cuta (wanda ya ƙunshi SDS/NaOH).

Buffer P4:neutralizing buffer.

Endotoxin Scavenger:yadda ya kamata cire endotoxin daga danyen plasmid tsantsa.

Buffer PW:wanke buffer, ƙara ƙayyadaddun ƙarar ethanol kafin amfani da farko.

Buffer TB:buffer buffer.

FastPure DNA Maxi ginshiƙai:ginshiƙan adsorption DNA na plasmid.

Tubu mai tarin 50 ml:tacewa tarin bututu.

Tube Tarin Kyauta mara Endotoxin:plasmid DNA tarin tubes.

Shirye-shiryen Kayayyakin

Absolute ethanol, isopropanol, 50 ml zagaye-ƙasa centrifuge tubes da 50 ml endotoxin-freecentrifuge tubes.

Aikace-aikace

Wannan samfurin ya dace da babban sikelin hakar plasmids daga 150 - 300 ml na maganin kwayan cuta.al'ada na dare.

Tsarin Gwaji

1. Ɗauki 150 - 200 ml (ba fiye da 300 ml ba) na maganin ƙwayoyin cuta da aka al'ada na dare da kuma centrifuge a.kimanin 11,000 rpm (12,000 × g) na 1 - 2 min.Yi watsi da abin da ke sama kuma a tattara kwayoyin cuta.

∆ Lokacin tattara fiye da 50 ml na maganin ƙwayoyin cuta, ana iya tattara ƙwayoyin cuta ta hanyar ƙara maganin ƙwayoyin cuta, centrifugation, zubar da supernatant da sauran matakai a cikin bututun 50 ml iri ɗaya don

sau da yawa.

2. Ƙara 7.5 ml na Buffer P1 (don Allah a duba ko an ƙara RNaseA zuwa Buffer P1) zuwa centrifugebututu mai dauke da kwayoyin cuta kuma a gauraya sosai ta vortex ko pipetting.

∆ Cikakkiyar dawowar ƙwayoyin cuta a cikin wannan matakin yana da mahimmanci don samar da albarkatu, kuma kada a sami ƙumburi na kwayan cuta bayan dawowar.Idan akwai ƙwayoyin ƙwayoyin cuta waɗanda ba a haɗa su sosai ba, zai shafi lysis, yana haifar da ƙananan yawan amfanin ƙasa da tsabta.Idan OD600 na maganin ƙwayar cuta shine 0.65, ana bada shawarar cewa a yi amfani da 7.5 ml na Buffer P1 lokacin cirewa daga 150 ml na maganin ƙwayar cuta;lokacin da OD600 shine 0.75, 8 ml na Buffer P1 yakamata a yi amfani da shi kuma ya kamata a canza kundin Buffers P2 da P4 daidai.Idan an ƙara ƙarar maganin ƙwayar cuta zuwa 200 ml, ana bada shawarar cewaƙarar Buffers P1, P2, da P4 za a ƙara daidai gwargwado.

3. Ƙara 7.5 ml na Buffer P2 zuwa dakatarwar kwayan cuta daga mataki na 2 kuma haɗuwa a hankali sama da ƙasa don 6 - 8sau da yawa a cikin dakin da zafin jiki na 4 - 5 min.

∆ Juya a hankali don haɗawa sosai.Vortexing zai haifar da rarrabuwar DNA na genomic, wanda zai haifar da gutsuttsuran DNA a cikin plasmid da aka fitar.A wannan lokacin, maganin ya zama danko kuma yana nunawa, yana nuna cewa kwayoyin sun kasance cikakke.Tsawon lokacin bai kamata ya wuce 5 min ba don guje wa lalata plasmids.Idan maganin bai bayyana ba, ana iya samun ƙwayoyin cuta da yawa da ke haifar da sulysis bai cika ba, don haka ya kamata a rage adadin ƙwayoyin cuta yadda ya kamata.

4. Ƙara 7.5 ml na Buffer P4 zuwa dakatarwar kwayan cuta daga mataki na 3 kuma nan da nan juya a hankali sau 6 - 8 don ba da damar maganin ya kawar da Buffer P2 gaba daya.A wannan lokacin, farin flocculent hazo ya kamata ya bayyana.Centrifuge a fiye da 11,000 rpm (12,000 × g) na 10 - 15 min, a hankali ya zubar da maɗaukaki cikin sabon bututu mai zagaye na ƙasa na 50 ml (wanda aka shirya da kansa), kuma a guje waaspirate da farin hazo.

∆ Ƙara Buffer P4 kuma nan da nan juya don haɗawa da kyau.Bar bututun ya tsaya har sai an rarraba farar hazo a ko'ina cikin maganin don hana samar da hazo na gida wanda zai iya shafar tsaka tsaki.Idan babu wani uniform farin flocculent hazo kafin centrifugation kuma supernatant bai bayyana bayan centrifugation, da bututu iya zama.centrifuged na wani 5 min.

5. Ƙara 0. Sau 1 na ƙarar (10% na girman girman girman, kimanin 2.2 ml) na Endotoxin Scavenger zuwa maɗaukaki daga mataki na 4 kuma juya zuwa haɗuwa.Sanya maganin a cikin wanka na kankara ko saka cikin kankara da aka niƙa (ko injin daskarewa) na 5 min har sai bayani ya canza daga turbid zuwa bayyananne da bayyane (ko har yanzudan kadan turbid), kuma lokaci-lokaci Mix sau da yawa.

∆ Bayan an ƙara Scavenger na Endotoxin zuwa maɗaukaki, mai ƙarfi zai zama turbid ammasupernatant ya kamata ya zama bayyananne (ko dan turbid) bayan sanyaya a cikin wankan kankara.

6. Bayan an sanya supernatant a dakin da zafin jiki (> 25 ℃) don 10 - 15 min, zai zama turbid kamar yaddazafinta yana ƙaruwa zuwa yanayin zafi.Sa'an nan kuma ya kamata a juya mai girma zuwa gauraye.

∆ Idan zafin dakin ya yi ƙasa ko kuna son rage lokacin cirewa, za'a iya shigar da supernatant a cikin ruwan wanka na 37 ~ 42 ℃ na tsawon mintuna 5 - 10 kuma ana iya aiwatar da mataki na gaba bayan supernatant.ya zama turbid.

7. Centrifuge da supernatant a game da 11,000 rpm (12,000 × g) na 10 min a dakin zafin jiki (zazzabi dole ne> 25 ℃) don raba lokaci.Tsarin ruwa na sama yana ƙunshe da DNA yayin da ƙaramin shuɗi mai launin shuɗi ya ƙunshi endotoxin da sauran ƙazanta.Canja wurinHalin ruwa mai ɗauke da DNA zuwa sabon bututu dajefar da m Layer.

∆ Zazzabi a lokacin centrifugation dole ne ya wuce 25 ℃ kamar yadda ingantaccen lokaci rabuwa bayafaruwa idan yanayin zafi yayi ƙasa sosai.

Δ Idan rabuwar lokaci ba ta da tasiri, ana iya daidaita zafin jiki na centrifugation zuwa 30 ℃ dalokacin centrifugation za a iya ƙara zuwa 15 min.

∆ Kar a tsotsi ruwan mai mai shudi saboda yana dauke da endotoxin da sauran datti.

Makanikai

Resuspension Lysis Neutralization

◇ Ƙara 7.5 ml Buffer P1

◇ Ƙara 7.5 ml Buffer P2

◇ Ƙara 7.5 ml Buffer P4

Cirewar Endotoxin

◇ Ƙara 0. Sau 1 mafi girman girman Endotoxin Scavenger

Daure da Wankewa

◇ Ƙara sau 0.5 na isopropanol

◇ Ƙara 10 ml Buffer PW