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==Procedure== ===Prepare Enzyme Dilutions=== *We want to dilute the supermutant-Tn5 and EZ-Tn5 **EZ-Tn5 provided in 5x concentration, so dilute using the following: 0.25ulA + 0.25ulB + 9.5ul water **supermutant-Tn5 provided in 25x concentration, so dilute using the following: 0.25ulA + 0.25ulB + 49.5ul water *We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 2ul ProtQ + 200ul Water ===Protocol=== *Prep tagmentation mastermix (will do 5.5 reactions for each strip tube as described above) 1x 5.5x (ul) Template 1 -- Nxt 1 5.5 <- either EZ-Tn5 or supermutant HMW Buff 1 5.5 Water 2 11 '''Make sure to make two different mastermixes (one for EZ-Tn5 and one for supermutant)''' *Incubate: 55C 5min -> remove tubes and put on ice block *Add 1 ul 1/100d ProtQ to each reaction *Incubate: 50C 10min -> 70C 20min -> 10C forever *Prep Exo- mastermix (add 1ul to each rxn) 1x 10.5x (ul) 10U/ul Exo- 0.1 1.05 25mM dNTP 0.4 4.2 Water 0.5 5.25 *Incubate: 37C 15min -> 75C 20min -> 10C forever *Add the following i5 adaptor (S5xx, containing index 2) and 1ul i7 adaptor (N7xx, containing index 1) in the following order. This is based off of the Nextera protocol recommendations for low plexity reactions found on [[File:cw_2015_02_26_Nextera_DNA_Sample_Prep_Guide.pdf]] **'''Note: Originally I thought I used N505/N506 for the index two primers, but ends up I really used S505/S506. This is because the indices are actually based off of the Nextera XT kit as found on [[File:2015_03_31_Nextera_xt_sample_preparation_guide.pdf]]. The corrections can be seen below''' rxn # Indx 1 (i7) Indx 2 (i5) 1A N701 (TAAGGCGA) S505 (GTAAGGAG) 2A N702 (CGTACTAG) "" 3A N703 (AGGCAGAA) "" 4A N704 (TCCTGAGC) "" 5A N705 (GGACTCCT) "" ---------------------------------------------- 1B N701 (TAAGGCGA) S506 (ACTGCATA) 2B N702 (CGTACTAG) "" 3B N703 (AGGCAGAA) "" 4B N704 (TCCTGAGC) "" 5B N705 (GGACTCCT) "" *Prep PCR mastermix -> add 11ul to each rxn 1x 10.5x KAPA SYBR Fast (2x) 10 105 Water 1 10.5 *Incubate: 72C 3min -> [98C 10sec -> 63C 30sec -> 72C 3min]x13 -> 10C *Purify using Ampure beads at 0.8:1 bead:sample ratio. Elute in 20ul H2O *Run 3ul on TBE gel *Purify again (same ratio) before sequencing if low-weight fragments appear on gel
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