TaqMan® probe functioning While carrying out a TaqMan® experiment, a fluorogenic probe, complementary to the target sequence is added to the PCR reaction mixture. This probe is an oligonucleotide with a reporter dye attached to the 5' end and a quencher dye attached to the 3' end. Till the time the probe is not hydrolized, the quencher and the fluorophore remain in proximity to each other, separated only by the length of the probe. This proximity however, does not completely quench the flourescence of the reporter dye and a background flourescence is observed. During PCR, the probe anneals specifically between the forward and reverse primer to an internal region of the PCR product. The polymerase then carries out the extension of the primer and replicates the 29 template to which the TaqMan® is bound. The 5' exonuclease activity of the polymerase cleaves the probe,
B rakke iseloomustab CD19, NK rakke CD16 ja CD56. Need on pinnamolekulid, mille abil on võimalik klasse eristada. Selleks kasutatakse monoklonaalseid antikehi. Meie lisasime kahte antikeha – CD-le saab eristada teistest rakkudest CD14 retseptoritega. Kõikide leukotsüütide pinnal on CD45. Definitsioon (FACS). Osakesed viiakse vedeliku laminaarse voolu abil tsütomeetri sisse detekteerimse punkti. Rakud peavad olema vedelikus. FACS – flourescence-activated cell sorting. Koosneb – voolutusosast, optilisest osast (genereerib laseritega valgussignaale), elektroonika osa – võimendab signaale, konverteerib neid ja salvestab. Nõel, mis ulatub katseklaasi. Selle abil detekteerimispunkti. Sinna sattunud osake murrab, hajutab valgust. Osa aga peegeldab organellidelt ja graanulitelt tagasi – seda osa nimetatakse side scatter e SSC. Mida rohkem on osakese sees graanuleid, seda suurem on side scatter
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