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==Top48 Hexamer RT Primer in vitro Validation: Nextera Tagmentation== *Continued from: [[Matt:LabNotes/2014-6-9]] *Since running a gel without digesting RNA (eg with RNase A) can't confirm success of RT, must continue with Nextera, and successful tagmentation indicates successful RT and Second Strand Synthesis ===Protocol=== *Taken from [[Arichard:Protocols/MDA_amplicon_tagmentation_and_amplification_2013_09_05 | Andrew's protocol]] ====Tagmentation==== # Add 2 ul HMW buffer to each PCR tube # Transfer 7ul of each sample to tubes #*PosCtrl: 1.06ng/ul #*NegCtrl: 0.884ng/ul #*Experimental: 0.818ng/ul #*NexteraControl (gDNA): 0.803ng/ul # Add 1 ul transposase (1:50 dilution) to each sample. Incubated 5.5 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 ====PCR with adapters and primers==== *6-17-2014 # 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. #*PosCtrl: Indx 26 #*NegCtrl: Indx 27 #*Exp: Indx 28 #*gDNA: Indx 29 # 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:2014-06-17_Top48HexPrimer_NexteraPCR.jpg | 250px]] # 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:2014-06-17_RTPrimerinvitroTest_Nextera.jpg | 250px]] <!--# 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. Proceed directly to size selection or store at 4 degC.--> ===Conclusion=== *Negative Control had double stranded DNA somehow nullifying the experiment
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