Kutshanje, ndifumene into emangalisayo!Uninzi lweengcali zovavanyo oluphambili olumjikelezileyo aziwazi namanqaku olwazi olusisiseko sovavanyo.

Ngokomzekelo, ngaba ungayiphendula le mibuzo ilandelayo?

Ngaba kukho umahluko phakathi kwe-OD260 kunye ne-A260?Ithetha ukuthini nganye?
I-OD sisishunqulelo se-optical density (i-optical density), A sisishunqulelo se-absorbance (absorbance), iikhonsepthi ezimbini ziyafana ngokwenene, "uxinzelelo lwe-optical" ngu "absorbance", kodwa "ubuninzi bokukhanya" buhambelana nemigangatho emininzi yelizwe kunye nemigangatho engaphezulu.

Ngokuqhelekileyo silinganisa ixabiso le-OD kwi-260nm ukubala i-nucleic acid concentration, ngoko i-1OD imele ntoni?
I-Nucleic acid inomlinganiselo ophezulu wokufunxa kwi-wavelength ye-260nm, equlethe zombini i-DNA kunye ne-RNA, kunye namaqhekeza e-nucleic acid (le ngongoma ephambili).
Ixabiso le-OD elilinganiswe kwi-wavelength ye-260 nm ibhalwe njenge-OD260.Ukuba isampuli ihlambulukile, ixabiso le-OD260 lingakwazi ukubala ukuxinwa kwesampuli ye-nucleic acid.
1 OD260=50 μg/ml dsDNA (i-DNA enemisonto emibini)
=37 μg/ml ssDNA (iDNA enemisonto enye)
=40 μg/ml RNA
=30 μg/ml dNTPs (oligonucleotides)
Ngaba kukho naluphi na unxibelelwano kunye nomahluko phakathi kwe-RT-PCR, iRealtime-PCR kunye neQPCR?
I-RT-PCR imfutshane ye-Reverse Transcription PCR
Ixesha lokwenyani PCR=qPCR, lifutshane kwi-Quantitative Real Time PCR
Nangona i-Reverse Transcription PCR (reverse transcription PCR) zombini zibonakala zifinyeziwe njenge-RT-PCR.Kodwa indibano yezizwe ngezizwe yile: I-RT-PCR ibhekisa ngokuthe ngqo kwi-PCR eshicilelweyo ebuyela umva.

Zeziphi eziqhele ukusetyenziswa nt, bp, kunye kb ukuchaza ubude DNA/RNA kwibhayoloji?
nt = i-nucleotide
bp = isiseko sesibini sesibini
kb = kilobase

Kakade ke, ubuya kuthi abantu abaninzi abazikhathaleli ezi nkcukacha zincinane!Wonke umntu uyakwenza oku, kwaye akukho mntu uya kukubuza ukuba yintoni na.Uyazi ukuba oku akuyomfuneko, akunjalo?

Hayi, Hayi, Hayi, kubaluleke kakhulu ukwazi oku!ngenxa yantoni?
Kuba ufuna ukuthumela inqaku!Ubhuti!Nokuba ujonge ukuphumelela okanye ulandela impumelelo yophando lwesayensi, kufuneka uthembele kumanqaku ukuze uthethe!

Ukukhutshwa kwe-Nucleic acid kufuneka kube yeyona nto ilula kwaye isisiseko sovavanyo.Umgangatho we-nucleic acid extraction unquma ngokuthe ngqo iziphumo zeemvavanyo ezilandelayo.

Nangona senditshilo amaxesha amaninzi, basebaninzi abahlobo abangakhathaliyo.Ngesi sihlandlo ndagqiba kwelokuba ndilishiye inqaku!

Ubuncinci boLwazi lokuPapashwa koMfuniselo we-PCR weXesha eliNina, elibizwa ngokuba yi-MIQE, yiseti yemigaqo yovavanyo yobungakanani be-fluorescence esungulwe kumazwe ngamazwe, ephakamisa ubuncinci bemigangatho yolwazi lovavanyo oluyimfuneko ekuvavanyeni imifuniselo yobungakanani be-fluorescence yobungakanani bePCR kunye namanqaku apapashiweyo.Ngokusebenzisa iimeko zovavanyo kunye neendlela zokuhlalutya ezibonelelwe ngumvavanyi, abaphononongi banokuvavanya ngcono ukuqinisekiswa kweskimu sovavanyo lomphandi.

Ingabonwa ukuba kwicandelo le-nucleic acid extraction, ezi zinto zilandelayo zokubona ziye zacetywa,

U-“E” ubonisa iinkcukacha emazinikwe kwaye u-“D” ubonisa ulwazi olufanele ukubonelelwa xa kukho imfuneko.

Ifom inzima kakhulu, enyanisweni, ndifuna ukuthetha ukuba wonke umntu kufuneka aqale ukusuka

ubunyulu (D), isivuno (D), imfezeko (E) kunye nokungaguquguquki (E) ukuvavanya i-Nucleic acids kule miba mine.

Ngokwemikhwa yokulinga, okokuqala thetha malunga neendlela zokuvavanya ubunyulu kunye nokugxila.

Umlinganiselo we-OD yeyona ndlela ithandwayo nelula yokufumanisa yabenza umfuniselo.Ngokuphathelele umgaqo, andiyi kungena kwiinkcukacha apha.Iilabhoratri ezininzi ngoku zisebenzisa i-ultra-micro spectrophotometers ukuhlalutya ngokuthe ngqo ngokobungakanani iisampulu ze-nucleic acid.Ngelixa ubonisa ixabiso lokufunxa, inkqubo inika ngokuthe ngqo ixabiso lokugxininiswa (i-nucleic acid, iprotheni kunye nedayi ye-fluorescent) kunye nemilinganiselo ehambelanayo.Ngokuphathelele uhlalutyo lwexabiso le-OD, Gcina lo mfanekiso kwaye uya kulunga.

Uluhlu lwesisombululo sexabiso le-OD ye-Universal

Nangona kunjalo, kukho izilumkiso ezimbalwa ekufuneka zikhutshwe ngokwahlukeneyo kuwe.

(Emva kwayo yonke loo nto, ndiyazi ukuba kufuneka ibe ngabo abagcina imali kwaye balinde de ubafune!)

Qaphela i-1 yezixhobo

Ixabiso le-OD liya kuchaphazeleka kwizixhobo ezahlukeneyo.Ngethuba nje i-OD260 ingaphakathi kuluhlu oluthile, amaxabiso e-OD230 kunye ne-OD280 anentsingiselo.Ngokomzekelo, uluhlu lwe-absorbection ye-Eppendorf D30 eqhelekileyo kwi-260nm ngu-0 ~ 3A, kunye ne-NanoDrop One ye-Thermo kwi-260nm.Uluhlu lokuthatha i-0.5 ~ 62.5A.

Inqaku lesi-2I-reagent ye-dilution

Ixabiso le-OD linokuchaphazeleka ngokuxutywa kwama-reagents ahlukeneyo.Ngokomzekelo, ukufundwa kwe-OD260/280 ye-RNA ecocekileyo kwi-pH7.5 10mM Trisisithinteli siphakathi kwe-1.9-2.1, ngelixa ingaphakathiisisombululo samanzi angathathi hlangothiumlinganiselo uya kuba ngaphantsi, mhlawumbi kuphela 1.8-2.0, kodwa oku akuthethi ukuba umgangatho RNA utshintsho Difference.

Inqaku lesi-3Izinto ezishiyekileyo

Ubukho bezinto ezishiyekileyo buya kuchaphazela ukuchaneka komlinganiselo woxinaniso lwe-nucleic acid, ngoko ke kuyimfuneko ukuphepha iiprotheyini, i-phenol, i-polysaccharide kunye neentsalela ze-polyphenol kwiisampuli ze-nucleic acid kangangoko kunokwenzeka.

Nangona kunjalo, enyanisweni, ukutsalwa nge-organic reagents yindlela yakudala.Kwiikiti zorhwebo, umphumo wokukhupha unokuphunyezwa ngekholamu ye-adsorption esekelwe kwi-silica edibeneyo kunye ne-centrifugation, ukuphepha i-reagents ezinobuthi ezinobuthi kunye nezinobungozi okunzima ukuzisusa, njl.Ikhithi yokutsalwa kwe-nucleic acid ye-Foregene, ayisebenzisi i-DNase/RNase kunye ne-organic reagents enetyhefu kulo lonke ixesha lokusebenza, ngokukhawuleza nangokukhuselekileyo., kwaye iisiphumo sikulungile(ngengozi yathi yimpandla, kodwa ndiyazi ukuba ufuna ukwazi).

Umzekelo 1: Imveliso ye-Genomic DNA extraction kunye nobunyulu

I-Foregene Soil DNA Isolation Kit (DE-05511) iphatha iisampulu zomhlaba kwimithombo eyahlukeneyo, kwaye isixa kunye nokusulungeka kwe-DNA ye-genomic efunyenweyo iboniswe kolu luhlu lulandelayo:
Umzekelo 2: Imveliso ye-RNA yokutsalwa kwezicubu kunye nobunyulu

I-Animal Total RNA Isolation Kit (RE-03012) esetyenzisiweyo iisampulu zeethishu ezahlukeneyo, kwaye isixa kunye nokusulungeka kwe-RNA efunyenweyo kuboniswe kwitheyibhile engezantsi (yezicubu zempuku):
Nangona kunjalo, sukucinga ukuba uyenziwe ngexabiso le-OD.Ingaba unawo nawaphi na amanyathelo abalulekileyo endikuzobele wona ngaphambili?

Qaphela

Iimolekyuli ze-nucleic acid eziqhekezayo nazo ziya kubalwa kwi-absorpension.Ukucinga ukuba uneentsalela ze-DNA ze-genomic kwi-RNA, ixabiso lakho le-OD liya kubonakala liphezulu kakhulu, kodwa i-concentration yangempela ye-RNA ayinakuqinisekiswa.Ingaba i-RNA yakho Akukacaci ukuba kukho ukuthotywa, ngoko sisafuna indlela yokuvavanya ebanzi ukunika isigwebo esichanekileyo, oko kukuthi, uvavanyo lwengqibelelo ye-nucleic acid ekhankanywe kwi-MIQE.