Bzalani Direct PCR Kit

Nambala ya mphaka: HCR2020A

Plant Direct PCR Kit ndi yoyenera kukulitsa mwachindunji masamba a zomera, mbewu, ndi zina zotero, ndipo ingagwiritsidwe ntchito powunika kwambiri zitsanzo za zomera zomwe zilibe ma polysaccharides ndi polyphenols.Kukulitsa mwachindunji DNA polymerase kutengera kusinthika komwe kumayendetsedwa kumakhala ndi kulolerana kopambana kwa PCR inhibitors muzomera.Pakadali pano, imasunga magwiridwe antchito apamwamba, omwe ndi oyenera kukulitsa zidutswa za DNA mkati mwa 5 kb.Malo apadera a Lysis buffer A mu zida atha kugwiritsidwa ntchito kusungunula minyewa yatsopano kapena yozizira.Ndiosavuta kugwiritsa ntchito ndipo lysate imatha kugwiritsidwa ntchito ngati template yokulitsa popanda kuyeretsedwa.Dongosololi lili ndi zoteteza zomwe zimapangitsa kuti zitsanzo zopanda pake zikwezedwe bwino pambuyo pozizira komanso kusungunuka mobwerezabwereza.2 × Plant Direct Master Mix imangofunika kuwonjezera zoyambira ndi ma templates kuti zigwirizane ndi matalikidwe, potero kuchepetsa magwiridwe antchito a mapaipi ndikuwongolera kuzindikira ndi kutulutsanso zotsatira.

Zigawo

*Plant Direct Lysis Buffer B ndi njira yosinthira, yomwe imagwiritsidwa ntchito kuletsa Plant Direct Lysis Buffer A kuti italikitse nthawi yosungiramo zitsanzo.Itha kugwiritsidwa ntchito molingana ndi momwe zilili.

Zosungirako

2 × Plant Direct Master Mix, sungani pa -30 ~ -15 ℃ ndipo pewani kuzizira mobwerezabwereza ndi kusungunuka;Bzalani Direct Lysis Buffer, sungani pa -30 ~ -15 ℃ kapena 2 ~ 8 ℃.

Njira Yoyesera

Zitsanzo Processing-Tsamba la Chomera

Njira yolunjika:Ndi bwino kugwiritsa ntchito masamba achinyamata.Gwiritsani ntchito nkhonya ya dzenje yokhala ndi mainchesi okhazikika a 0.5 - 3 mm kuti mupeze samplea yaying'ono komanso yofananira, ndiyeno yonjezerani mwachindunji chitsanzocho ku dongosolo la PCR (50 μl system ikulimbikitsidwa).Zindikirani, onetsetsani kuti chitsanzocho chili mu njira ya PCR osati motsutsana ndi khoma la chubu.Ngati PCR yolunjika imagwiritsidwa ntchito kukulitsa zidutswa zazitali ndi zitsanzo zovuta, pogwiritsa ntchito chitsanzo chokhala ndi mainchesi ang'onoang'ono (0.5 - 1 mm) monga template ingathandize kupeza zotsatira zabwino.

Njira yopangira lysis:Ndi bwino kugwiritsa ntchito masamba achinyamata.Tengani tsamba laling'ono (pafupifupi 1 - 3 mm m'mimba mwake), liyikeni mu 20 μl Plant Direct Lysis Buffer Ab, ndipo perani momwe mungathere ( sitepe iyi ikhoza kuchitika pofinya tsambalo ndi nsonga ya pipette 100 μl. phatikizani chitsanzo).Ngati masamba okulirapo agwiritsidwa ntchito (osapitirira 7 mm), onjezani kuchuluka kwa dilution buffer mpaka 50 μl.Masamba akatha, yankho liyenera kuwoneka lobiriwira.Pambuyo pa centrifugation mwachidule, onjezani 1 μl ya supernatant ku PCR system monga reaction templatec.

Thermal lysis njira:Ndi bwino kugwiritsa ntchito masamba achinyamata.Tengani katsamba kakang'ono (pafupifupi 1 - 3 mm m'mimba mwake), ikani mu 20 μl Plant Direct Lysis Buffer A, ndi kutentha pa 95 ° C kwa 5 - 10 min.Nthawi ya lysis imatha kukulitsidwa moyenera kwa masamba omwe ndi ovuta kutulutsa (osapitirira 20 min).Ngati masamba okulirapo agwiritsidwa ntchito (osapitirira 7 mm), onjezani kuchuluka kwa dilution buffer mpaka 50 μl.Mukatenthetsa, ikani centrifuge mwachidule, ndikuwonjezera 1 μl ya supernatant ku PCR system ngati templateb reaction.

Zitsanzo Processing-Bzalani Mbewu

Njira yopangira lysis:Gwiritsani ntchito scalpel kudula njere ndi mainchesi 5 mm, kuwonjezera pa 100 μl ya Plant Direct Lysis Buffer A, ndikupera nyembazo ndi nsonga ya pipette kapena zida zina.Vortex mwachidule ndikuyimirira kutentha kwa 3 - 5 min.Onetsetsani kuti mbeuyo yamizidwa mu dilution buffer.Pambuyo pa centrifugation mwachidule, onjezani 1 μl ya supernatant ku PCR system ngati template yochitira.

Thermal lysis njira:Gwiritsani ntchito scalpel kudula njere zokhala ndi mainchesi 5 mm, onjezerani ku 100 μl wa Plant Direct Lysis Buffer A, ndi kutentha pa 95 ° C kwa 5 - 10 min.Nthawi ya lysis imatha kukulitsidwa moyenera kwa masamba omwe ndi ovuta kutulutsa (osapitirira 30 min).Mukatenthetsa, ikani centrifuge mwachidule, ndikuwonjezera 1 μl supernatant ku PCR system ngati templateb reaction.

a.Lumo kapena zida zina zitha kugwiritsidwanso ntchito podula zitsanzo za kukula koyenera;ngati nkhonya kapena lumo agwiritsidwanso ntchito, ayenera kutsukidwa ndi 2% sodium hypochlorite solution musanagwiritse ntchito kuti mupewe kuipitsidwa pakati pa zitsanzo.

b.Onetsetsani kuti Plant Direct Lysis Buffer yasungunuka kwathunthu musanagwiritse ntchito.Ngati chotchinga chili ndi viscous kapena chili ndi mpweya, chimatha kutenthedwa pa 37 ℃ kuti chisungunuke kwathunthu musanagwiritse ntchito.

c.Voliyumu ya template mu dongosolo anachita akhoza kusinthidwa moyenera malinga ndi kusiyana kwa buku la zinthu zomera ndi diluent anawonjezera.

Bzalani Direct Lysis Buffer

The Plant Direct Lysis Buffer A yomwe ili mu mankhwalawa idakonzedwa bwino kuti itulutse ma genome amitundu yambiri yazimera ndipo ndiyoyenera kusungidwa kwakanthawi kochepa kwa zomera zopanda 4 ℃.Ngati chitsanzocho chiyenera kusungidwa kwa nthawi yotalikirapo (mwachitsanzo, 1 - 2 miyezi), tikulimbikitsidwa kusamutsa supernatant ku chubu chatsopano cha EP ndikusunga pa -20 ℃.Kuti musunge zitsanzo mokhazikika, onjezerani voliyumu yofanana ya Plant Direct Lysis Buffer B ku champhamvu chomwe chasinthidwa, sakanizani bwino ndikusunga pa -20 ℃.Nthawi yosungirako yokhazikika imasiyanasiyana ndi zitsanzo za zomera ndi mayiko.

Reaction System

a.Muli ndi Mg²⁺pamlingo womaliza wa 2 mM.

b.Ndibwino kuti mugwiritse ntchito ndende yomaliza ya 0.4μM pa chiyambi chilichonse.Kugwiritsa ntchito kwambiri ma primers kumapangitsa kuti kuchuluke mosadziwika bwino.

c.Kuchuluka kwa zitsanzo zomwe zimagwiritsidwa ntchito zimatha kusinthidwa malinga ndi momwe zilili.The ndalama ntchito limodzi anachita ya yaiwisi lysed chitsanzo akhoza kusintha pakati pa 2% - 20% ya okwana buku la anachita.Kugwiritsa ntchito zitsanzo zambiri kungayambitse kulephera kwa amplification.

Pulogalamu ya Reaction

a.Denaturation Yoyamba (98 ℃, 5 min) imalimbikitsa lysis ya minofu ya zomera, kutulutsa DNA ya genomic yomwe ingagwiritsidwe ntchito kukulitsa PCR.Osafupikitsa nthawi kapena kuchepetsa kutentha.

b.Ndibwino kuti muyike yofanana ndi mtengo woyambira Tm kapena 2 ~ 4 ℃ kuposa mtengo wa Tm.Kukulitsa mwachindunji DNA polymerase yomwe imagwiritsidwa ntchito popanga izi ndi yosiyana ndi yamba ya Taq DNA polymerase, ndipo ili ndi zofunikira zapadera pa kutentha kwa kutentha; kugwiritsa ntchito kutentha kwapamwamba kumatha kuchepetsa kukulitsa kosafunikira ndikuwongolera magwiridwe antchito.Kwa ma tempuleti ovuta, kukulitsa koyenera kumatha kutheka mwa kusintha kutentha kwa annealing ndikutalikitsa nthawi yowonjezera.

c.Ngati kutalika kwa mankhwala okulitsa ndi ≤1 kb, nthawi yowonjezera imayikidwa pa 30 sec/kb;ngati kutalika kwa mankhwala okulitsa ndi> 1 kb, nthawi yowonjezera imayikidwa pa 60 sec/kb.

d.Kwa zitsanzo zovuta kapena zitsanzo zokhala ndi zokolola zochepa zokulitsa, chiwerengero cha maulendo chikhoza kuwonjezeka moyenerera mpaka 40 -50.

Mapulogalamu

Imagwiritsidwa ntchito pakukulitsa kwachindunji kwa minyewa yazomera ndikuwunika kwambiri zitsanzo za zomera zomwe zilibe ma polysaccharides ndi ma polyphenols.

Zolemba

Kugwiritsa ntchito kafukufuku kokha.Osagwiritsidwa ntchito pozindikira matenda.

1. Pakukula kwa mbewu zopanda pake kapena kukulitsa kwachindunji, tikulimbikitsidwa kugwiritsa ntchito purified genomic DNA ngati chiwongolero chabwino musanayambe kuyesa kuwonetsetsa kuti dongosolo, zoyambira ndi ntchito ndizolondola.

2. Kukweza molunjika kwa DNA polymerase yomwe imagwiritsidwa ntchito mu chida ichi imakhala ndi ntchito yowunikira.Ngati TA cloning ikuyenera kuchitidwa, tikulimbikitsidwa kuyeretsa DNA musanawonjezere adenine.

3. Upangiri Woyambira:

a.Ndikofunikira kuti maziko omaliza kumapeto kwa 3′ akuyenera kukhala G kapena C.

b.Kusagwirizana kotsatizana kuyenera kupewedwa m'magawo 8 omaliza kumapeto kwa 3′ koyambira.c.Pewani mapangidwe a hairpin kumapeto kwa 3 ′ koyambira.

d.Kusiyana kwa mtengo wa Tm woyambira kutsogolo ndi woyambira kumbuyo kuyenera kusapitilira 1 ℃ ndipo mtengo wa Tm uyenera kusinthidwa kukhala 60 ~ 72 ℃ (Primer Premier 5 ikulimbikitsidwa kuwerengera mtengo wa Tm).

e.Njira zowonjezera zowonjezera zomwe sizikugwirizana ndi template, siziyenera kuphatikizidwa powerengera mtengo wa Tm woyambira.

f.Ndikoyenera kuti GC zili mu primer kukhala 40% -60%.

g.Kugawidwa konse kwa A, G, C ndi T mu choyambirira kuyenera kukhala kofanana momwe kungathekere.Pewani kugwiritsa ntchito zigawo zomwe zili ndi GC kapena AT zambiri.

h.Pewani kukhalapo kwa zoyambira 5 kapena kupitilira apo kapena pakati pa zoyambira ziwiri ndipo pewani kupezeka kwa magawo atatu kapena kupitilira apo kumapeto kwa 3′ kwa zoyambira ziwiri.

ndi.Gwiritsani ntchito ntchito ya NCBI BLAST kuti muwone tsatanetsatane wa choyambira kuti mupewe kukulitsa mosadziwika bwino.