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====Adapter PCR==== #Finish first round PCR master mix (per reaction): ## 15 ul 2X KAPA fast SYBR. ## 0.6 ul Adapter 1. ## 1.4 ul H2O. # Add 0.6 of barcode adapters to each sample #*Random Primer Mix: 26 #*Oligo (dT)23 VN: 27 #*FISSEQ_RT: 28 #*Top48 Hexamer: 29 #*NTC: 30 #*No Enzyme: 31 #*NxtraPosControl (gDNA): 33 #*NxtraNegControl (H2O): 34 #*NxtraPCRControl (H2O): 35 # Add 17 ul of first round PCR master mix to each sample # Incubate in thermocycler: ## 95 degC for 1 min. ## 7 cycles: ### 95 degC for 10 sec. ### 62 degC for 15 sec. ### 72 degC for 45 sec. # Finish second round PCR master mix: ## 35 ul KAPA fast SYBR ## 2 ul Primer 1 ## 2 ul Primer 2 ## 31 ul H2O # Add 70 ul of second round PCR master mix to each sample. # PCR program is as follows: ## 95 degC for 1 min. ## 10 cycles ### 95 degC for 10 sec. ### 60 degC for 15 sec. ### 72 degC for 45 sec. # Incubate in thermocycler an additional 3 min @ 72 degC for final extension. [[File:20140929_RTprimertestNxtraPCR.JPG | 650px]] *RanHex, dT, FISSEQ_RT, Top48, and NxtraPosControl (gDNA) all had amplification as expected *NTC, NoEnzyme, NxtraNegControl, and NxtraPCRControl all had no amplification as expected **NoEnzyme had no amplification which is GOOD unlike last time, indicates all DNA was removed from UHRR
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