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=Nextera Lib Construction on CH-S-A/B/C/D= ==Background== *Previously, I tested the supermutant Tn5 on CH-S-A sea bacteria samples and found that the 0.25x concentration was best for the 1ng of input (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-2-27>). However, the positive control (using Nextera v1) did not work. After talking with Brandon, I realized that the reason why the positive control reaction didn't work was because I added the wrong primers to the PCR reaction. I used the new Nextera dual-indexing primers instead of the original Nextera single-indexing primers that we've used before. Without the correct primers, the adapter sequences were not recognized and that's why the PCR didn't work at all *Today, I'll be mostly repeating the same experiment as done on 2015_02_27, only this time, I'll be doing the correct reactions and also will be using all four of the CH-S samples to provide a more representative test for the library construction protocols. *I'll be assuming that the new Ez-Tn5 produces about the same activity as Nextera v1, so I'll be doing a 1/50 dilution of the enzyme prior to use *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 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 **-20C 3rd shelf Brandon's Tn5 box (contains the EZ-Tn5 and supermutant) *Below are the reactions we'll be using today: Rxn # Template Enzyme Enzyme Dilution 1A CH-S-A EZ-Tn5 0.25x 2A CH-S-B "" "" 3A CH-S-C "" "" 4A CH-S-D "" "" 5A --(NTC) "" "" -------------------------------------------------- 1B CH-S-A sm-Tn5 0.25x 2B CH-S-B "" "" 3B CH-S-C "" "" 4B CH-S-D "" "" 5B --(NTC) "" "" ==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 ==Results== *Below is the gel image for this experiment: (Low mass ladder, 1A->5A, x, 1B->5B) [[File:cw_ZhangLab_2_2015-03-10_16hr_34min_ez-tn5_and_supermutant_lib_construction_test.jpg|500px]] *Based on the gel image, we see that the libraries look good and we will continue with sequencing (perform paired end 150 sequencing on all 12 libraries from the Nextera experiments and the Illumina library prep experiments)
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