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=Nextera Lib Construction Test on Sea Bac (CH-S-A)= ==Background== *I have already made Illumina libraries from the four highest concentrated samples on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-2-18>. These four samples will also be used in a comparison test with two Nextera enzymes (original Nextera and supermutant): Concentration CH-S-A 63.2ng/ul CH-S-B 69.2 CH-S-C 83.9 CH-S-D 35.2 *We want to dilute the samples to ~1ng/ul using the following dilutions: Concentration ul Sample ul Water CH-S-A 63.2ng/ul 1 62 <- We will be using only this sample for today's test CH-S-B 69.2 1 68 CH-S-C 83.9 1 82 CH-S-D 35.2 1 34 *These libraries will be tested using the protocol provided by Andrew on <http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Protocols/tn5059>. The necessary reagents can be found in one of three places: **Andrew's 4C box **-20C 2nd shelf in Midas -20C box **-20C 2nd shelf in Nextera primer box *The Nextera supermutants are provided in the 50ml tubes labeled Control (the thick handwriting is 25x diluted enzyme) *For this test, we'll need to run several reactions: different dilutions of the enzyme (up to 1x dilution and below) and also include a control tube with the original Nextera enzyme (already diluted 1/50) *Below are the reactions and conditions we'll be testing today: Rxn # Template Enzyme Enzyme Dil 1 CH-S-A (1ng) Nxt Old 1/50 2 "" Nxt SM 1x 3 "" "" 0.75x 4 "" "" 0.50x 5 "" "" 0.25x 6 "" "" 0.1x 7 Water (NTC) Nxt Old 1/50 8 "" Nxt SM 1x ==Procedure== ===Prepare Enzyme Dilutions=== *We want to dilute the supermutant to the following dilutions (from the original 25x concentration): 1x 1ul supermutant + 11.5ul UV treated TE buff + 12.5ul glycerol 0.75x 3ul 1x dilution + 1ul Water 0.50x 2ul 0.75x dilution + 1ul Water 0.25x 1ul 0.50x dilution + 1ul Water 0.1x 1ul 1x dilution + 9ul Water *We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 0.2ul ProtQ + 200ul Water ===Protocol=== *Prep tagmentation mastermix (will do 8 reactions as described above) 1x 8.5x (ul) Template 1 -- Nxt 1 8.5 HMW Buff 1 8.5 Water 2 17 *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 8.5x (ul) 10U/ul Exo- 0.1 0.85 25mM dNTP 0.4 3.4 Water 0.5 4.25 *Incubate: 37C 15min -> 75C 20min -> 10C forever *Add the following i5 adaptor (N/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]] rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) N501 (TAGATCGC) 2 N702 (CGTACTAG) N502 (CTCTCTAT) 3 N703 (AGGCAGAA) N503 (TATCCTCT) 4 N704 (TCCTGAGC) N504 (AGAGTAGA) 5 N705 (GGACTCCT) N505 (GTAAGGAG) 6 N706 (TAGGCATG) N506 (ACTGCATA) 7 N707 (CTCTCTAC) N507 (AAGGAGTA) 8 N708 (CAGAGAGG) N508 (CTAAGCCT) *Prep PCR mastermix -> add 11ul to each rxn 1x 8.5x KAPA SYBR Fast (2x) 10 85 Water 1 8.5 *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 ==Results== *Below is the gel image for the experiment: (Low Mass Ladder, x, rxn's 1->8) [[File:cw_ZhangLab_2_2015-02-27_19hr_09min_Nextera_Lib_Construction_Test.jpg|500px]]
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