Cog direct PCR Kit
Cat No: HCR2020A
Plant Direct PCR Kit yog tsim rau kev nthuav dav ncaj qha ntawm cov nplooj, cov noob, thiab lwm yam, thiab tuaj yeem siv rau kev tshuaj ntsuam xyuas cov nroj tsuag uas tsis muaj polysaccharides thiab polyphenols.Qhov ncaj qha amplification DNA polymerase raws li cov lus qhia evolution muaj superior kam rau PCR inhibitors hauv cov nroj tsuag.Lub caij no, nws tuav cov kev ua haujlwm siab ua haujlwm siab, uas yog tsim rau kev nthuav dav ntawm DNA tawg hauv 5 kb.Qhov tshwj xeeb Lysis tsis nyob rau hauv cov khoom siv tuaj yeem siv los ua cov ntaub so ntswg tshiab lossis khov.Nws yooj yim rau kev khiav lag luam thiab cov lysate tuaj yeem siv los ua tus qauv rau kev nthuav dav yam tsis muaj kev ua kom huv.Lub kaw lus muaj cov tshuaj tiv thaiv uas ua rau cov qauv coj los ua kom muaj txiaj ntsig zoo tom qab rov ua kom khov thiab thawing.2 × Cog Direct Master Mix tsuas yog yuav tsum tau ntxiv primers thiab templates los ua cov tshuaj tiv thaiv amplification, yog li txo cov kev ua haujlwm pipetting thiab txhim kho kev tshawb nrhiav thiab kev tsim tawm ntawm cov txiaj ntsig.
Cheebtsam
| Cheebtsam | 50 NRX | 200 NRX |
| 2 × Cog Direct Master Mix | 1.25 ml ib | 4 × 1.25 ml |
| Cog Direct Lysis Buffer A | 5ml ib | 20 ml ib |
| Cog Direct Lysis Buffer B* | 5ml ib | 20 ml ib |
* Cog Direct Lysis Buffer B yog ib qho kev xaiv reagent, uas yog siv los neutralize Plant Direct Lysis Buffer A rau ncua sij hawm cia ntawm cov qauv.Nws tuaj yeem siv tau raws li qhov xwm txheej tiag tiag.
Kev cia khoom
2 × Cog Direct Master Mix, khaws cia ntawm -30 ~ -15 ℃ thiab tsis txhob rov khov thiab thawing;Cog Direct Lysis Buffer, khaws cia ntawm -30 ~ -15 ℃ lossis 2 ~ 8 ℃.
Kev sim txheej txheem
Qauv ua-Nplooj nplooj
Txoj kev ncaj nraim:Nws raug nquahu kom siv cov nplooj me me.Siv lub qhov punch nrog ib txoj kab uas hla ntawm 0.5 - 3 hli kom tau txais cov qauv me me thiab zoo ib yam, thiab tom qab ntawd ncaj qha ntxiv cov qauv mus rau PCR system (50 μl system raug pom zoo).Nco ntsoov, xyuas kom meej tias tus qauv yog nyob rau hauv cov tshuaj PCR thiab tsis tiv thaiv lub raj phab ntsa.Yog tias siv PCR ncaj qha los nthuav cov kab ntev ntev thiab cov qauv nyuaj, siv cov qauv nrog lub taub me me (0.5 - 1 mm) ua tus qauv tuaj yeem pab kom tau txais txiaj ntsig zoo dua.
Txoj kev sib tsoo lysis:Nws raug nquahu kom siv cov nplooj me me.Siv ib daim me me ntawm nplooj (li 1 - 3 hli inch), muab tso rau hauv 20 μl Plant Direct Lysis Buffer Ab, thiab zom nws kom ntau li ntau tau (cov kauj ruam no yuav ua tau los ntawm kev nyem cov nplooj nrog 100 μl pipette taub hau. mus mash tus qauv).Yog tias siv cov ntaub so ntswg loj dua (tsis pub tshaj 7 mm), ua kom lub ntim ntawm cov dej tsis haum rau 50 μl.Tom qab nplooj yog av, cov tshuaj yuav tsum tshwm ntsuab.Tom qab luv luv centrifugation, ntxiv 1 μl ntawm supernatant mus rau PCR system raws li cov tshuaj tiv thaiv templatec .
Thermal lysis method:Nws raug nquahu kom siv cov nplooj me me.Siv ib daim me me ntawm nplooj (li 1 - 3 hli inch), muab tso rau hauv 20 μl Cog Direct Lysis Buffer A, thiab kub ntawm 95 ° C rau 5 - 10 min.Lub sijhawm lysis tuaj yeem txuas ntxiv rau cov nplooj uas nyuaj rau lyse (tsis pub tshaj 20 min).Yog tias siv cov ntaub so ntswg loj dua (tsis pub tshaj 7 mm), ua kom lub ntim ntawm cov dej tsis haum rau 50 μl.Tom qab cua sov, centrifuge luv luv, thiab ntxiv 1 μl ntawm supernatant rau PCR system raws li cov tshuaj tiv thaiv templateb.
Qauv ua– Nroj tsuag
Txoj kev sib tsoo lysis:Siv lub scalpel txiav cov noob nrog txoj kab uas hla ntawm 5 hli, ntxiv rau 100 μl ntawm Cog Direct Lysis Buffer A, thiab zom cov qauv nrog lub taub hau lossis lwm yam cuab yeej.Vortex luv luv thiab cia sawv ntawm chav sov li 3 - 5 min.Xyuas kom tseeb tias cov noob qauv yog submerged nyob rau hauv lub dilution buffer.Tom qab luv luv centrifugation, ntxiv 1 μl ntawm supernatant mus rau PCR system raws li cov qauv tshuaj tiv thaiv.
Thermal lysis method:Siv lub scalpel txiav cov noob nrog txoj kab uas hla ntawm 5 hli, ntxiv rau 100 μl ntawm Cog Direct Lysis Buffer A, thiab kub ntawm 95 ° C rau 5 - 10 min.Lub sijhawm lysis tuaj yeem txuas ntxiv rau cov nplooj uas nyuaj rau lyse (tsis pub tshaj 30 min).Tom qab cua sov, centrifuge luv luv, thiab ntxiv 1 μl supernatant rau PCR system raws li cov tshuaj tiv thaiv templateb.
a.txiab lossis lwm yam cuab yeej siv tau los txiav cov qauv uas tsim nyog;yog tias cov punch lossis txiab rov qab siv dua, lawv yuav tsum tau ntxuav nrog 2% sodium hypochlorite tov ua ntej siv los tiv thaiv kev sib kis ntawm cov qauv.
b.Xyuas kom tseeb tias tsob nroj Direct Lysis Buffer yog tag nrho yaj ua ntej siv.Yog tias qhov tsis muaj dej khov lossis muaj nag lossis daus, nws tuaj yeem ua kom sov ntawm 37 ℃ kom yaj tag nrho ua ntej siv.
c.Lub ntim ntawm template nyob rau hauv cov tshuaj tiv thaiv kab mob tuaj yeem hloov kho kom haum raws li qhov sib txawv ntawm qhov ntim ntawm cov khoom cog thiab diluent ntxiv.
Cog Direct Lysis Buffer
Cov nroj tsuag Direct Lysis Buffer A muaj nyob rau hauv cov khoom no tau nruj me ntsis optimized tso tawm cov genome ntawm feem ntau cov ntaub so ntswg thiab yog haum rau luv luv cia ntawm crude nroj tsuag ntawm 4 ℃.Yog tias tus qauv xav tau khaws cia rau lub sijhawm ntev (xws li 1 - 2 lub hlis), nws raug nquahu kom hloov cov supernatant mus rau lub raj EP tshiab thiab khaws cia ntawm -20 ℃.Txhawm rau khaws cov qauv kom ruaj khov, ntxiv qhov sib npaug ntawm Cov Tsob Ntoo Direct Lysis Buffer B rau qhov hloov pauv supernatant, sib tov zoo thiab khaws cia ntawm -20 ℃.Lub sijhawm khaws cia ruaj khov sib txawv nrog cov qauv cog thiab lub xeev.
Reaction System
| ddH ua2O | Rau 20.0 µl | 50.0 µl |
| 2 × Cog Direct Master Mixa | 10.0 l os | 25.0 ;ua |
| Primer 1 (10 µM) | 0,8l ua | 2.0l ua |
| Primer 2 (10 µM)b | 0,8l ua | 2.0l ua |
| Cog nplooj / crude extract qauv(Saib rau kev ua piv txwv) | 0.5-3 hli nplooj disc / x µl | 0.5-3 hli nplooj disc / x µl |
a.Nws muaj Mg2+ntawm qhov kawg concentration ntawm 2 mM.
b.Nws raug nquahu kom siv qhov kawg ntawm 0.4μM rau txhua tus primer.Kev siv cov primers ntau dhau yuav ua rau muaj kev ua kom tsis muaj zog ntxiv.
c.Tus nqi ntawm cov qauv siv tuaj yeem hloov kho raws li qhov xwm txheej tiag tiag.Tus nqi siv nyob rau hauv ib qho tshuaj tiv thaiv ntawm crude lysed qauv tuaj yeem hloov kho ntawm 2% - 20% ntawm tag nrho cov ntim ntawm cov tshuaj tiv thaiv.Kev siv cov qauv ntau dhau tuaj yeem ua rau kev ua kom tsis muaj zog.
Reaction Program
| Cov kauj ruam | Kub | Sijhawm |
| Initial Denaturation | 98 ℃ | 5 feeb |
| Denaturation | 95 ℃ | 10 sec |
| Annealing | 58 ~ 72 ℃ | 15 sec |
| Txuas ntxiv | 72 ℃ | 30 sec |
| Kawg Extension | 72 ℃ | 5 feeb |
a.Initial Denaturation (98 ℃, 5 min) txhawb nqa lysis ntawm cov ntaub so ntswg, tso genomic DNA uas yuav siv tau rau PCR amplification.Tsis txhob txo lub sijhawm lossis txo qhov kub.
b.Nws raug nquahu kom teem nws sib npaug rau tus nqi primer Tm lossis 2 ~ 4 ℃ siab dua tus nqi Tm.Qhov ncaj qha amplification DNA polymerase siv nyob rau hauv cov khoom no yog txawv los ntawm cov pa Taq DNA polymerase, thiab muaj tshwj xeeb yuav tsum tau rau cov tshuaj tiv thaiv annealing kub; kev siv ntawm siab annealing kub yuav ua tau zoo txo cov nonspecific amplification thiab txhim kho lub amplification efficiency.Rau cov qauv nyuaj, qhov ua kom muaj zog tuaj yeem ua tiav los ntawm kev kho qhov kub thiab txias thiab ncua sij hawm ncua sij hawm.
c.Yog tias qhov ntev ntawm cov khoom lag luam amplification yog ≤1 kb, lub sijhawm txuas ntxiv yog teem rau ntawm 30 sec / kb;Yog tias qhov ntev ntawm cov khoom lag luam amplification yog> 1 kb, lub sijhawm txuas ntxiv yog teem rau ntawm 60 sec / kb.
d.Rau cov qauv nyuaj lossis cov qauv uas tsis tshua muaj suab nrov, cov voj voog tuaj yeem tsim nyog nce mus txog 40 -50 cycles.
Daim ntawv thov
Nws muaj feem xyuam rau kev nthuav dav ncaj qha ntawm cov ntaub so ntswg thiab kev soj ntsuam siab ntawm cov qauv cog uas tsis muaj polysaccharides thiab polyphenols.
Sau ntawv
Rau kev tshawb fawb siv xwb.Tsis siv rau hauv cov txheej txheem kuaj mob.
1. Rau crude cog amplification los yog ncaj qha amplification, nws yog pom zoo kom siv purified genomic DNA raws li ib tug zoo tswj ua ntej pib qhov kev sim los xyuas kom meej tias lub system, primers thiab kev khiav hauj lwm yog lawm.
2. Qhov ncaj qha amplification DNA polymerase siv nyob rau hauv cov khoom no muaj zog proofreading ua si.Yog tias TA cloning yuav tsum tau ua, nws raug pom zoo kom ntxuav cov DNA ua ntej ntxiv cov adenine.
3. Primer Design Guide:
a.Nws raug pom zoo tias lub hauv paus kawg ntawm 3' kawg ntawm cov primer yuav tsum yog G lossis C.
b.Sib law liag tsis sib haum yuav tsum tau zam nyob rau hauv 8 lub hauv paus kawg ntawm 3′ kawg ntawm primer.c.Zam cov qauv plaub hau ntawm 3′ kawg ntawm cov primer.
d.Qhov sib txawv ntawm Tm tus nqi ntawm cov primer pem hauv ntej thiab rov qab primer yuav tsum tsis txhob ntau tshaj 1 ℃ thiab Tm tus nqi yuav tsum tau hloov mus rau 60 ~ 72 ℃ (Primer Premier 5 yog pom zoo kom xam tus nqi Tm).
e.Ntxiv cov txheej txheem primer ntxiv uas tsis sib haum nrog cov qauv, yuav tsum tsis txhob suav nrog thaum xam tus nqi primer Tm.
f.Nws raug pom zoo tias GC cov ntsiab lus ntawm primer yuav tsum yog 40% -60%.
g.Tag nrho kev faib ntawm A, G, C thiab T hauv primer yuav tsum yog raws li qhov ua tau.Tsis txhob siv cov cheeb tsam uas muaj GC siab lossis AT cov ntsiab lus.
h.Zam kom tsis txhob muaj qhov sib ntxiv ntawm 5 lossis ntau lub hauv paus nyob rau hauv cov primer lossis nruab nrab ntawm ob primers thiab tsis txhob muaj qhov sib ntxiv ntawm 3 lossis ntau lub hauv paus ntawm 3′ kawg ntawm ob primers.
i.Siv NCBI BLAST muaj nuj nqi los xyuas qhov tshwj xeeb ntawm cov primer los tiv thaiv kev ua kom tsis muaj zog.
