Zomwe Ziyenera Kuganiziridwa Popanga Zosakaniza za PCR?

Kuti zichitike bwino machulukitsidwe, m'pofunika kuti munthu anachita zigawo zikuluzikulu alipo mu ndende yolondola iliyonse kukonzekera. Kuphatikiza apo, ndikofunikira kuti palibe kuipitsidwa komwe kumachitika.

Makamaka pamene zochita zambiri ziyenera kukhazikitsidwa, zakhazikitsidwa pokonzekera otchedwa master mix m'malo mwa pipetting aliyense reagent padera mu chotengera chilichonse. Zosakaniza zokonzedweratu zilipo malonda, momwe zigawo zachitsanzo zokhazokha (zoyambira) ndi madzi zimawonjezeredwa. Kapenanso, kusakaniza kwa master kumatha kukonzedwa nokha. M'mitundu yonseyi, kusakaniza kumagawidwa ku chotengera chilichonse cha PCR popanda template ndipo sampuli ya DNA imawonjezedwa padera pamapeto.

Kugwiritsa ntchito kaphatikizidwe ka master kumakhala ndi zabwino zingapo: Choyamba, kuchuluka kwa masitepe amodzi kumachepetsedwa. Mwa njira iyi, chiopsezo cha zolakwika za ogwiritsa ntchito panthawi ya pipetting ndi chiopsezo cha kuipitsidwa kumachepetsedwa ndipo, ndithudi, nthawi imasungidwa. M'malo mwake, kulondola kwa mapaipi ndikwambiri, chifukwa ma voliyumu akulu amaperekedwa. Izi ndizosavuta kumvetsetsa poyang'ana deta yaukadaulo ya pipettes: Voliyumu yocheperako, ndiye kuti zopatuka zimakwera kwambiri. Mfundo yakuti zokonzekera zonse zimachokera ku chotengera chimodzi zimakhala ndi zotsatira zabwino pa homogeneity (ngati zimasakanizidwa bwino). Izi zimathandiziranso kuchulukitsa kwa zoyesererazo.

Pokonzekera kusakaniza kwa master, osachepera 10% voliyumu yowonjezera iyenera kuwonjezeredwa (mwachitsanzo ngati kukonzekera 10 kumafunika, kuwerengera pamaziko a 11), kotero kuti ngakhale chotengera chomaliza chidzadzaza bwino. Mwa njira iyi, (pang'ono) kulakwitsa kwa pipetting, ndi zotsatira za kutaya chitsanzo pamene dosing zotsukira munali njira akhoza kulipidwa. Zotsukira zili munjira za ma enzyme monga ma polymerase ndi zosakaniza zazikulu, zomwe zimapangitsa kupanga thovu ndi zotsalira mkati mwazabwinobwino.malangizo a pipette.

Kutengera kugwiritsa ntchito komanso mtundu wamadzimadzi operekedwa, njira yoyenera yoperekera mapaipi (1) iyenera kusankhidwa ndikusankha zida zoyenera. Pamayankho omwe ali ndi zotsukira, njira yosinthira mwachindunji kapena nsonga zotchedwa "otsika posungira" nsonga za pipette monga m'malo mwa ma pipette a air-cushion akulimbikitsidwa. Zotsatira zaMalangizo a ACE PIPETTEzachokera makamaka hydrophobic pamwamba. Zamadzimadzi zomwe zili ndi zotsukira sizimasiya filimu yotsalira mkati ndi kunja, kotero kuti kutayika kwa yankho kungathe kuchepetsedwa.

Kupatula mlingo weniweni wa zigawo zonse, m'pofunikanso kuti kuipitsidwa kwa kukonzekera kumachitika. Sikokwanira kugwiritsa ntchito zogwiritsidwa ntchito zoyera kwambiri, chifukwa ndondomeko ya pipette mu mpweya wodutsa pipette imatha kupanga ma aerosol omwe amakhalabe mu pipette. DNA yomwe ingakhale mu aerosol ikhoza kusamutsidwa kuchokera ku chitsanzo chimodzi kupita ku yotsatira mu sitepe yotsatira ya pipetting ndipo motero imayambitsa kuipitsidwa. Njira zachindunji zosamuka zomwe zatchulidwa pamwambapa zithanso kuchepetsa ngoziyi. Pakuti mpweya khushoni pipettes n'zomveka ntchito fyuluta nsonga kuteteza chulucho pipette ndi kusunga splashes, aerosols, ndi biomolecules.