5× Neoscript Mai sauri RT-qPCR Premix da-UNG

Saukewa: HCB5142A

Neoscript Fast RT Premix-UNG (Probe qRT-PCR) ingantaccen haɗin tushen binciken bututu ne wanda ya dace da jujjuya juzu'i na mataki ɗaya da PCR (qRT-PCR).Yana goyan bayan haɗawa da abubuwan ƙira da bincike kuma ya tsaya barga bayan adana lokaci mai tsawo a ƙananan zafin jiki.Za'a iya ƙara samfurin da za a gwada kai tsaye lokacin amfani, ba tare da ƙarin aikin buɗawa / bututun bututu ba.Wannan samfurin yana ba da abubuwan haɗin gwiwa, misali DNA polymerase mai farawa, M-MLV, uracil DNA glycosylase (TS-UNG), Mai hana RNase, MgCl.₂, dNTPs (tare da dUTP maimakon dTTP), da stabilizers.Tare da gyaggyara saurin haɓakawa da juzu'i na juzu'i da DNA polymerase, yana yiwuwa a kammala haɓakar PCR a cikin mintuna 20-40.Wannan reagent yana amfani da buffer na musamman don qPCR tare da gauraye enzymes na anti-inhibitory amplification enzyme da UNG enzyme.Sabili da haka, yana iya samun ingantaccen haɓakar ƙwayoyin halittar da aka yi niyya kuma ya hana haɓakar ƙarar ƙarya ta hanyar ragowar PCR da gurɓataccen iska.Wannan reagent ya dace da yawancin kayan aikin PCR masu kyalli daga masana'antun kamar Applied Biosystems, Eppendorf, Bio-Rad da Roche.

Bangaren

1.25× Neoscript Fast RTase/UNG Mix

2.5 × Neoscript Fast RT Premix Buffer (dUTP)

Yanayin Ajiya

Duk abubuwan da aka gyara yakamata a kiyaye su a -20 ℃ don ajiya na dogon lokaci da 4 ℃ har zuwa watanni 3.Da fatan za a haxa sosai bayan narke da centrifuge kafin amfani.Guji daskarewa akai-akai.

qRT-PCR Shirye-shiryen Tsarin Amsa

1) a. Ƙarshen ƙaddamarwa na farko shine yawanci 0.2μM.Don ingantacciyar sakamako, za'a iya inganta maida hankali a cikin kewayon 0.2-1μM.Gabaɗaya, ana iya inganta ƙaddamar da binciken a cikin kewayon 0.1-0.3μM.

2) b.Lokacin da ake amfani da Tsarin PCR mai sauri, haɓaka ƙaddamarwa na farko da bincike na iya haifar da sakamako mafi kyau na haɓakawa, kuma ya kamata a inganta rabonsu daidai.

3) Nau'in samfurori daban-daban sun ƙunshi nau'o'in nau'i daban-daban da abun ciki na mai hanawa da kuma kwafin adadin kwayoyin halitta.Ya kamata a yi la'akari da ƙarar samfurin ta ainihin yanayin.Yi dilution na samfurin tare da ruwan da ba shi da nuclease ko TE Buffer, idan ya cancanta.

Ra'ayin Cal'amura

Kula da inganci

1.Gano aikin: hankali, ƙayyadaddun ƙayyadaddun ƙayyadaddun aiki da maimaitawar qPCR.

2.Babu aikin nuclease na waje: babu exogenous endonuclease da exonuclease gurbatawa.

Bayanan kula

1.Ƙimar haɓakawa na DNA polymerase mai sauri bai wuce 1kb/10s ba.Kayan aikin PCR daban-daban suna da saurin dumama da sanyaya daban-daban, yanayin sarrafa zafin jiki da haɓakar zafin jiki, don haka haɓaka ƙaddamarwar firamare/binciken ku da hanyar gudu tare da takamaiman kayan aikin PCR ɗinku mai sauri yana da mahimmanci.

2.Wannan samfurin yana aiwatar da fa'ida mai fa'ida, kuma ya dace da babban ganewar ƙwayoyin ƙwayoyin cuta.Ana ba da shawarar hanyar PCR mai matakai uku don masu farawa waɗanda ke da ƙarancin zafin jiki ko don haɓaka dogon gutsuttsura sama da 200 bp.

3.Tunda amplicons daban-daban suna da tasirin amfani daban-daban na dUTP da kuma hankali daban-daban ga UNG, yakamata a inganta reagents idan ƙwarewar ganowa ta ragu yayin amfani da tsarin UNG.Da fatan za a tuntuɓe mu don tallafin fasaha idan an buƙata.

4.Don guje wa haɓaka samfuran PCR masu ɗaukar nauyi, ana buƙatar yanki na gwaji da pipette don haɓakawa.Yi aiki da safar hannu kuma canza akai-akai kuma kar a buɗe bututun PCR bayan haɓakawa.