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==Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR== *Must first remove dsDNA from UHRR since there was contamination [[Matt:LabNotes/2014-6-18 | last time]] ===Purify UHRR of DNA=== *Use Zymo DNA-Free RNA Kit *Sample: 4ul of UHRR (1.3 ug/ul) ====DNase I Digest==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | UHRR (1.3ug/ul)||4 |- | 10X DNase I Buffer||5 |- | DNase I||2 |- | H2O||39 |- | Total||50 |} *Mix and incubate at 37C for 30min ====Purify==== *Add 100ul RNA Binding Buffer and mix *Add 150ul 100% EtOH and mix *Transfer to Zymo-Spin IC Column in Collection Tube **Centrifuge at 13,000rcf for 1min and discard flow through *Add 400ul RNA Prep Buffer **Centrifuge at 13,000rcf for 1min and discard flow through *Add 800ul RNA Wash Buffer **Centrifuge at 13,000rcf for 30sec and discard flow through *Add 400ul RNA Wash Buffer **Centrifuge at 13,000rcf for 30sec and discard flow through *Centrifuge at 13,000rcf for 2min and discard flow through *Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min **Centrifuge at 10,000rcf for 30sec *Store at -20C ===Reverse Transcription & Second Strand Synthesis=== ====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 |- | Purified UHRR||1||Purified UHRR||1||Purified UHRR||1||Purified UHRR||1||Purified UHRR||0||Purified UHRR||1 |- | 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||H2O||5||H2O||6||H2O||6||H2O||6||H2O||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 ====Nextera Tagmentation==== # Add 2 ul HMW buffer to each PCR tube (8 total) # Transfer 7ul of each sample to tubes #*Random Primer Mix #*Oligo (dT)23 VN #*FISSEQ_RT #*Top48 Hexamer #*NTC #*No Enzyme #*NxtraPosControl (gDNA): 0.803 ng/ul #*NxtraNegControl (H2O) # 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): 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 ====Bead Purification==== # Add 100 ul magnetic beads to each positive sample (samples 1-4). 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 ====Gel Size Selection==== *Ladder: 0.5ul Low Mass Ladder + 2.5ul H2O + 2ul Loading Dye *Samples: 5ul Sample + 2ul Loading Dye *Gel: [[File:2014-10-03_RTprimerTestSizeSelect.jpg | 450px]] #Selected size: 200-800bp #Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. #Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. #Added 450 ul of 1X TE buffer #Vortexed for 60 min at 37 C in incubator #Centrifuged at 15,000 rpm for 3 min at RT #Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min #Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min #Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube) #Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 #Vortexed and placed the 4 tubes at -80C for overnight #Spun 4 tubes in -80C at 10,000rpm at 4C for 30min #Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min #Discard supernatant and let dry in hood 10 min #Resuspended each of the 4 tubes with 10ul H2O
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