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==Repeat Top48 Hexamer RT Primer in vitro Validation== ===Procedure=== ====First Strand Synthesis==== *Mix RNA in Primer and H2O *Denature for 5 minutes at 70C **Put on ice right after *Add Reaction Mix and Enzyme *Incubate for 5 min at 25C *Incubate for 1 hour at 42C *Inactivate enzyme for 5 min at 80C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Random Primer Mix''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Oligo d(T)23 VN''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''FISSEQ_RT''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Top48 Hexamer''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''NTC''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''No Enzyme''' | align="center" style="background:#f0f0f0;"|'''''' |- | Components||Volume||Components||Volume||Components||Volume||Components||Volume||Components||Volume||Components||Volume |- | 1.3ug/ul UHRR||0.5||1.3ug/ul UHRR||0.5||1.3ug/ul UHRR||0.5||1.3ug/ul UHRR||0.5||1.3ug/ul UHRR||0||1.3ug/ul UHRR||0.5 |- | Random Primer Mix (60uM)||2||Oligo d(T)23 VN (50uM)||2||FISSEQ_RT (100uM)||1||Top48 Hexamer (100uM)||1||Random Primer Mix (60uM)||2||Random Primer Mix (60uM)||2 |- | H2O||5.5||H2O||5.5||H2O||6.5||H2O||6.5||H2O||6||H2O||5.5 |- | Denature Step||||Denature Step||||Denature Step||||Denature Step||||Denature Step||||Denature Step|| |- | M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10 |- | M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||H2O||2 |} ====Second Strand Synthesis==== *Add 48 ul H20 *Add 8ul 10x Second strand synthesis reaction buffer *Add 4ul Second strand synthesis enzyme mix *Mix by pipetting *Incubate for 2.5 hours at 16C ====Purification & Measure Concentration==== *Qiagen PCR Purification **Elute with 30ul *Nanodrop quant: **Random Primer Mix: 15.2 ng/ul **Oligo (dT)23 VN: 23.6 ng/ul **FISSEQ_RT: 9.7 ng/ul **Top48 Hexamer: 24.3 ng/ul **NTC: 14.5 ng/ul **No Enzyme: 25.0 ng/ul *Negative controls (NTC & No Enzyme) did not show significantly less DNA, could be RNA absorbing light and "fooling" the Nanodrop *Actual concentrations probably much less since previous comparison with Qubit showed [[Matt:LabNotes/2014-6-5#Check_Concentration | 25x less]] ====Nextera Tagmentation==== # Add 2 ul HMW buffer to each PCR tube (8 total) # Transfer 7ul of each sample to tubes #*Random Primer Mix: 15.2 ng/ul #*Oligo (dT)23 VN: 23.6 ng/ul #*FISSEQ_RT: 9.7 ng/ul #*Top48 Hexamer: 24.3 ng/ul #*NTC: 14.5 ng/ul #*No Enzyme: 25.0 ng/ul #*NxtraPosControl (gDNA): 0.803 ng/ul #*NxtraNegControl (H2O): 0 ng/ul # Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5 minutes at 55 degC # Dilute Qiagen protease 1:100 in H2O. Add 1 ul to each sample and incubate 10 minutes at 50 degC, followed by 20 minutes at 70 degC # Make the master mix for Exo- treatment: 1 ul Exo- and 0.4 ul dNTP per reaction # Add 1.4 ul Exo- mix to each sample and incubate 15 minutes at 37 degC, followed by 20 minutes at 75 degC ====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): 32 #*NxtraNegControl (H2O): 33 #*NxtraPCRControl (H2O): 34 # 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:06202014_Top48HexPrimer_NxtraPCRtrial2.JPG | 650px]] # Run 5 ul sample on PAGE gel with 3 ul 6X loading dye to identify positives. ## 0.5 ul low mass ladder (Invitrogen). Equalize volume with TBE. ## 25 min @ 250 V. ## Stain 5-10 min in ~100 ml 1X TBE with 6 ul SYBR Safe. [[File:2014-06-20_RTPrimerinvitroTest_Nextera_trial2.jpg | 650px]] # Add 100 ul magnetic beads to each positive sample. Mix by pipetting and incubate 8 minutes off magnet # Place on magnet 5 minutes, then remove and discard supernatant # On magnet, wash 2x with 180 ul 80% EtOH. Let each wash sit 30 seconds removing # Let dry 5 minutes # Resuspend beads off magnet with 20 ul H2O and incubate 2 minutes, then return to magnet for 1 minute. # Extract supernatant and transfer to 0.2 ml tube *Continued on: [[Matt:LabNotes/2014-6-23]]
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