Enweghị mgbama nkwalite

1.Taq DNA Polymerase na ngwa ahụ na-efunahụ ọrụ ya n'ihi nchekwa na-ezighị ezi ma ọ bụ njedebe nke ngwa ahụ.
Nkwanye: Kwado ọnọdụ nchekwa nke ngwa ahụ;tinyeghachi Taq DNA Polymerase kwesịrị ekwesị na sistemu PCR ma ọ bụ zụta ngwa PCR Real Time ọhụrụ maka nnwale ndị metụtara ya.

2.E nwere ọtụtụ ihe mgbochi nke Taq DNA Polymerase na DNA template.
Aro: Megharịa ndebiri ma ọ bụ belata ọnụọgụ ndebiri ejiri.

3.The Mg2 + ịta adịghị adabara.
Nkwanye: Mg2 + ntinye nke the2 × Real PCR Mix anyị na-enye bụ 3.5mM.Agbanyeghị, maka ụfọdụ primers na ndebiri pụrụ iche, ntinye Mg2+ nwere ike ịdị elu.Ya mere, ị nwere ike tinye MgCl2 ozugbo iji bulie mkpokọta Mg2+.A na-atụ aro ka ịbawanye Mg2+ 0.5mM oge ọ bụla maka njikarịcha.

4.The PCR amplification ọnọdụ adịghị adabara, na primer usoro ma ọ bụ itinye uche na-ekwesịghị ekwesị.
Aro: gosi izi ezi nke usoro nke primer ma emebiela primer;Ọ bụrụ na mgbama nkwuwapụta adịghị mma, gbalịa iwetulata okpomọkụ na-ekpo ọkụ ma mezie ntinye nke primer nke ọma.

5. Ọnụ ọgụgụ nke template dị ntakịrị ma ọ bụ nke ukwuu.
Nkwanye: Mee template linearization gradient dilution, wee họrọ mkpokọta template nwere mmetụta PCR kacha mma maka nnwale PCR oge.

NTC nwere oke fluorescence uru

1.Reagent mmetọ mere n'oge ọrụ.
Nkwanye: Dochie na ọhụrụ reagents maka ezigbo oge PCR nnwale.

2.Contamination mere n'oge nkwadebe nke usoro mmeghachi omume PCR.
Nkwanye: Were usoro nchebe dị mkpa mgbe ị na-arụ ọrụ, dị ka: iyi uwe latex, iji ntu pipette nwere nzacha, wdg.

3.The primers na-eweda n'ala, na mmebi nke primers ga-eme ka ndị na-abụghị kpọmkwem amplification.
Aro: Jiri SDS-PAGE electrophoresis chọpụta ma ọ̀tụ̀tụ̀tụ̀ ihe ndị e ji emepụta ihe na-emebi emebi, ma jiri primers ọhụrụ dochie ha maka nnwale PCR Real Time.

Primer dimer ma ọ bụ nkwalite na-abụghị nke akọwapụtara

1.The Mg2 + ịta adịghị adabara.
Nkwanye: Mg2 + ntinye uche nke 2 × Real PCR EasyTM Mix anyị na-enye bụ 3.5 mM.Agbanyeghị, maka ụfọdụ primers na ndebiri pụrụ iche, ntinye Mg2+ nwere ike ịdị elu.Ya mere, ị nwere ike tinye MgCl2 ozugbo iji bulie mkpokọta Mg2+.A na-atụ aro ka ịbawanye Mg2+ 0.5mM oge ọ bụla maka njikarịcha.

2.The PCR annealing okpomọkụ dị oke ala.
Aro: Mee ka PCR annealing okpomọkụ site 1℃ ma ọ bụ 2℃ oge ọ bụla.

3.The PCR ngwaahịa dị ogologo.
Nkwanye: Ogologo ngwaahịa PCR Real Time kwesịrị ịdị n'etiti 100-150bp, ọ bụghị karịa 500bp.

4.The primers na-eweda n'ala, na nbibi nke primers ga-eduga na ọdịdị nke kpọmkwem mmụba.
Aro: Jiri SDS-PAGE electrophoresis chọpụta ma ọ̀tụ̀tụ̀tụ̀ ihe ndị e ji emepụta ihe na-emebi emebi, ma jiri primers ọhụrụ dochie ha maka nnwale PCR Real Time.

5.The PCR usoro na-ekwesịghị ekwesị, ma ọ bụ usoro dị oke obere.
Aro: Sistemụ mmeghachi omume PCR pere mpe ga-eme ka nchọta nke ọma belata.Ọ kacha mma iji usoro mmeghachi omume nke akụrụngwa PCR ọnụọgụ tụrụ aro iji megharịa nnwale PCR Real Time.

Nkwughachi nke ụkpụrụ ọnụọgụgụ adịghị mma

1. The ngwá bụ malfunctioning.
Aro: Enwere ike inwe mperi n'etiti oghere PCR ọ bụla nke ngwaọrụ ahụ, na-ebute mmeghari na-adịghị mma n'oge njikwa okpomọkụ ma ọ bụ nchọpụta.Biko lelee dị ka ntuziaka nke ngwa kwekọrọ.

2.The sample ịdị ọcha adịghị mma.
Nkwanye: Ihe nlele na-adịghị ọcha ga-eduga na mmeghari nke nnwale ahụ na-adịghị mma, nke gụnyere ịdị ọcha nke template na primers.Ọ kacha mma imegharị ndebiri ahụ, yana SDS-PAGE na-asachapụ ihe ndị ahụ.

3.The PCR usoro nkwadebe na oge nchekwa dị ogologo.
Aro: Jiri ezigbo oge PCR usoro maka PCR nnwale ozugbo nkwadebe, na ahapụla ya n'akụkụ ruo ogologo oge.

4.The PCR amplification ọnọdụ adịghị adabara, na primer usoro ma ọ bụ itinye uche na-ekwesịghị ekwesị.
Aro: gosi izi ezi nke usoro nke primer ma emebiela primer;Ọ bụrụ na mgbama nkwuwapụta adịghị mma, gbalịa iwetulata okpomọkụ na-ekpo ọkụ ma mezie ntinye nke primer nke ọma.

5.The PCR usoro na-ekwesịghị ekwesị, ma ọ bụ usoro dị oke obere.
Aro: Sistemụ mmeghachi omume PCR pere mpe ga-eme ka nchọta nke ọma belata.Ọ kacha mma iji usoro mmeghachi omume nke akụrụngwa PCR ọnụọgụ tụrụ aro iji megharịa nnwale PCR Real Time.