cDNA Synthesis_AuPre Gold Kit
c-DNA Synthesis:
· Using Oligo(dt)20 Primer
o Thaw each tube in the AuPreP Gold cDNA Synthesis Kit and place on ice
o Briefly centrifuge all reagents componenets
o Reaction Mix
§ RNA (1.2µg) - 2µl
§ Primer (2µM) - 1µl
§ Water - 7µl
o Mix gently and heat the mixture at 700C for 5min. Remove tube and place rapidly on ice
o Add following components to the reaction tube:
§ Reaction Mix (2.5X) - 8µl
§ DTT (100mM) - 2µl
o Mix gently by flicking the tube, centrifuge briefly
o Incubate the reaction tube at 420C for 60min
o Heat at 700C for 15min, centrifuge the mix briefly
o Use for PCR and store at -200C
· Using Random Hexamer Primers
o Thaw each tube in the AuPreP Gold cDNA Synthesis Kit and place on ice
o Briefly centrifuge all reagents componenets
o Reaction Mix
§ RNA (1.2µg) - 2µl
§ Primer (2µM) - 1µl
§ Water - 7µl
o Mix gently and heat the mixture at 700C for 5min. Remove tube and place rapidly on ice
o Add following components to the reaction tube:
§ Reaction Mix (2.5X) - 8µl
§ DTT (100mM) - 2µl
o Mix gently by flicking the tube, centrifuge briefly
o Incubate the reaction tube at 370C for 60min
o Heat at 700C for 15min, centrifuge the mix briefly
o Use for PCR and store at -200C
· Using Specific Primer
o Thaw each tube in the AuPreP Gold cDNA Synthesis Kit and place on ice
o Briefly centrifuge all reagents componenets
o Reaction Mix
§ RNA (1.2µg) - 2µl
§ Primer F (2µM) - 0.5µl
§ Primer R (2µM) - 0.5µl
§ Water - 7µl
o Mix gently and heat the mixture at 700C for 5min. Remove tube and place rapidly on ice
o Add following components to the reaction tube:
§ Reaction Mix (2.5X) - 8µl
§ DTT (100mM) - 2µl
o Mix gently by flicking the tube, centrifuge briefly
o Incubate the reaction tube at 420C for 60min
o Heat at 700C for 15min, centrifuge the mix briefly
o Use for PCR and store at -200C
· Control cDNA Synthesis
o Thaw each tube in the AuPreP Gold cDNA Synthesis Kit and place on ice
o Briefly centrifuge all reagents componenets
o Reaction Mix
§ Control RNA (1µg) - 1µl
§ Primer (2µM) - 1µl
§ Water - 8µl
o Mix gently and heat the mixture at 700C for 5min. Remove tube and place rapidly on ice
o Add following components to the reaction tube:
§ Reaction Mix (2.5X) - 8µl
§ DTT (100mM) - 2µl
o Mix gently by flicking the tube, centrifuge briefly
o Incubate the reaction tube at 420C for 60min
o Heat at 700C for 15min, centrifuge the mix briefly
o Use for PCR and store at -200C