Muva nje, ngithole okuthile okumangalisayo!Ochwepheshe abaningi bokuhlola okuthuthukile abaseduze kwakhe abazi ngisho namaphuzu athile olwazi lokuhlola ayisisekelo.

Ngokwesibonelo, ungayiphendula yini imibuzo elandelayo?

Ingabe ukhona umehluko phakathi kwe-OD260 ne-A260?Isho ukuthini ngayinye?
I-OD isifinyezo se-optical density (i-optical density), A isifinyezo se-absorbance (absorbance), imiqondo yomibili iyafana ngempela, “ukuminyana kokubona” kusho “ukungafinyeleli”, kodwa “ukuminyana kokubona” kuhambisana nezindinganiso eziningi zikazwelonke futhi kusezingeni eliphezulu.

Ngokuvamile sikala inani le-OD ku-260nm ukuze sibale ukugxiliswa kwe-nucleic acid, ngakho-ke imele ini i-1OD?
I-Nucleic acid inenani eliphakeme lokumunca kubude begagasi obungu-260nm, obuqukethe kokubili i-DNA ne-RNA, kanye nezingcezu ze-nucleic acid ezihlukene (leli iphuzu elibalulekile).
Inani le-OD elikalwa kubude begagasi obungu-260 nm laqoshwa njenge-OD260.Uma isampuli imsulwa, inani le-OD260 lingakwazi ukubala ukugxiliswa kwesampula ye-nucleic acid.
1 OD260=50 μg/ml dsDNA (i-DNA enemicu ekabili)
=37 μg/ml ssDNA (i-DNA enomucu owodwa)
=40 μg/ml i-RNA
=30 μg/ml dNTPs (oligonucleotides)
Ingabe kukhona ukuxhumana nomahluko phakathi kwe-RT-PCR, Realtime-PCR ne-QPCR?
I-RT-PCR imfushane ye-Reverse Transcription PCR
Isikhathi Sangempela PCR=qPCR, esifushane se-Quantitative Real Time PCR
Nakuba i-Reverse Transcription PCR (Reverse Transcription PCR) zombili zibonakala zifushaniswa njenge-RT-PCR.Kodwa umhlangano wamazwe ngamazwe uthi: I-RT-PCR iqondise ngokuqondile ku-PCR yokubhala kabusha.

Yiziphi i-nt, bp, ne-kb ezivame ukusetshenziswa ukuchaza ubude be-DNA/RNA kubhayoloji?
nt = i-nucleotide
bp = i-base pair base pair
kb = kilobase

Yebo, ungasho ukuthi abantu abaningi abanandaba nale mininingwane emincane!Wonke umuntu uyakwenza lokhu, futhi akekho ozokubuza ukuthi kuyini.Uyazi ukuthi lokhu akudingekile, akunjalo?

Cha, Cha, Cha, kubaluleke kakhulu ukwazi lokhu!ngenxa yani?
Ngoba ufuna ukuthumela isihloko!Mfowethu!Noma ngabe uhlose ukuthweswa iziqu noma uphishekela impumelelo yocwaningo lwesayensi, kufanele uthembele kuma-athikili ukuze ukhulume!

Ukukhishwa kwe-Nucleic acid kufanele kube isilingo esilula nesiyisisekelo.Ikhwalithi yokukhishwa kwe-nucleic acid inquma ngokuqondile imiphumela yokuhlolwa okulandelayo.

Nakuba sengikusho kaningi kodwa basebaningi abangani abangenandaba.Kulokhu nganquma ukuphuma kulesi sihloko!

Ulwazi Oluncane Lokushicilelwa Kokuhlolwa Kwesikhathi Sangempela Kwe-PCR, okubizwa nge-MIQE, isethi yemihlahlandlela yokuhlolwa komthamo we-fluorescence eyethulwe emazweni ngamazwe, ephakamisa amazinga amancane olwazi lokuhlola oludingekayo ukuze kuhlolwe ukuhlolwa komthamo we-fluorescence kwe-PCR nama-athikili okushicilela.Ngezimo zokuhlola nezindlela zokuhlaziya ezinikezwe umhloli, ababuyekezi bangahlola kangcono ukufaneleka kohlelo lokuhlola lomcwaningi.

Kungabonakala ukuthi esigabeni sokukhishwa kwe-nucleic acid, izinto ezilandelayo zokutholwa ziye zahlongozwa:

U-“E” ubonisa ulwazi okufanele luhlinzekwe futhi u-“D” ubonisa ulwazi okufanele luhlinzekwe uma kunesidingo.

Ifomu liyinkimbinkimbi kakhulu, empeleni, ngifuna ukusho ukuthi wonke umuntu udinga ukuqala kusuka

ubumsulwa (D), isivuno (D), ubuqotho (E) nokuvumelana (E) ukuze kuhlolwe ama-Nucleic acid kulezi zici ezine.

Ngokwemikhuba yokuhlola, okokuqala khuluma ngezindlela zokuhlola zokuhlanzeka nokugxila.

Ukulinganisa kwe-OD kuyindlela eyintandokazi nelula yokuthola yabahloli.Ngokuqondene nomgomo, ngeke ngingene emininingwaneni lapha.Amalabhorethri amaningi manje asebenzisa ama-ultra-micro spectrophotometer ukuze ahlaziye ngokuqondile amasampula e-nucleic acid.Ngenkathi ibonisa inani lokumunca, uhlelo lunikeza ngokuqondile inani lokugxilisa ingqondo (i-nucleic acid, amaprotheni nodayi we-fluorescent) kanye nezilinganiso ezihlobene.Ngokuqondene nokuhlaziywa kwevelu ye-OD, Londoloza lesi sithombe futhi uzolunga.

Uhlu lwesixazululo senani le-OD ye-Universal

Kodwa-ke, kunezixwayiso ezimbalwa okufanele zikhishwe ngokwehlukana kwakho.

(Phela, ngiyazi ukuthi kufanele kube yibona abagcina imali bese nilinda nize nibadinge!)

Qaphela 1 Izinsiza

Inani le-OD lizothinteka amathuluzi ahlukene.Uma nje i-OD260 ingaphakathi kwebanga elithile, amanani we-OD230 kanye ne-OD280 abalulekile.Isibonelo, ububanzi bokumunca be-Eppendorf D30 evamile ku-260nm ngu-0~3A, kanti i-NanoDrop One ye-Thermo iku-260nm.Ibanga lokumunca lingu-0.5~62.5A.

Inothi 2I-reagent ye-dilution

Inani le-OD lingathinteka ngokuhlanjululwa kwama-reagents ahlukene.Isibonelo, ukufundwa kwe-OD260/280 kwe-RNA ehlanzekile ku-pH7.5 10mM I-Trisisigcinalwazi siphakathi kuka-1.9-2.1, ngenkathi singaphakathiisixazululo samanzi esingathathi hlangothiisilinganiso sizoba ngaphansi, mhlawumbe kuphela i-1.8-2.0, kodwa lokhu akusho ukuthi izinga le-RNA lishintsha Umehluko.

Inothi 3Izinto ezisele

Ukuba khona kwezinto ezisele kuzothinta ukunemba kwesilinganiso sokuhlushwa kwe-nucleic acid, ngakho-ke kuyadingeka ukugwema amaprotheni, i-phenol, i-polysaccharide nezinsalela ze-polyphenol kumasampuli e-nucleic acid ngangokunokwenzeka.

Kodwa-ke, empeleni, ukukhishwa ngama-organic reagents kuyindlela endala.Emakhithini ezentengiselwano, umphumela wokukhipha ungafinyelelwa ngekholomu ye-adsorption esekelwe ku-silica ehlanganiswe ne-centrifugation, ukugwema ama-reagents anobuthi nayingozi okunzima ukuwasusa, njll. Inkinga, njengeIkhithi yokukhipha i-nucleic acid ye-Foregene, ayisebenzisi i-DNase/RNase nama-organic organic reagents anobuthi phakathi nokusebenza, ngokushesha futhi kuphephile., kanye neumphumela uwukuthikuhle( ngephutha wathi impandla, kodwa ngiyazi ufuna ukwazi).

Isibonelo sokuqala: Isivuno sokukhishwa kwe-Genomic DNA nobumsulwa

I-Foregene Soil DNA Isolation Kit (DE-05511) iphatha amasampula omhlabathi avela emithonjeni ehlukahlukene, futhi inani nokuhlanzeka kwe-DNA ye-genomic etholiwe kukhonjisiwe kuthebula elilandelayo:
Isibonelo sesi-2: Isivuno sokukhishwa kwezicubu ze-RNA nobumsulwa

I-Animal Total RNA Isolation Kit (RE-03012) icubungule amasampula ezicubu ezihlukahlukene, futhi inani nokuhlanzeka kwe-RNA etholiwe kuboniswa kuthebula elingezansi (lezicubu zegundane):
Nokho, ungacabangi ukuthi usuqedile ngevelu ye-OD.Ingabe unawo amaphuzu abalulekile engikudwebele wona ngaphambili?

Qaphela

Ama-molecule e-nucleic acid ahlukanisiwe nawo azobalwa ekumunceni.Uma sicabanga ukuthi unezinsalela ze-DNA ye-genomic ku-RNA, inani lakho le-OD lizobonakala liphezulu kakhulu, kodwa ukugxila kwangempela kwe-RNA akukwazi ukunqunywa.Ukuthi i-RNA yakho Akukacaci ukuthi kukhona ukuwohloka, ngakho-ke sisadinga indlela yokuhlola ebanzi ukuze sinikeze ukwahlulela okunembe kakhudlwana, okungukuthi, ukuhlolwa kobuqotho be-nucleic acid okukhulunywe ngayo ku-MIQE.