Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31
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Nextera Lib Construction on Catalina Harbor Samples[edit]
Background[edit]
- Previously, I tested the supermutant Tn5 on samples CH-S-A/B/C/D and found that the 0.25x concentration worked the best for 1ng of input (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-10>
- Today, I'll be performing library construction using the supermutant Tn5 on the remaining Catalina Harbor samples, which were all >100ng total yield.
- We want to dilute the samples to ~1ng/ul using the following dilutions:
Concentration ul Sample ul Water Test 1 5.22ng/ul 1 4.22 Test 2 5.09 1 4.09 CH-P-A 6.67 1 5.67 CH-P-B 18.8 1 17.8 CH-P-C 14.1 1 13.1 CH-P-D 13.6 1 12.6 CH-O-A (Filter 1) 12.6 1 11.6 CH-O-B (Filter 2) 27.6 1 26.6
- The necessary reagents can be found in one of four 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 1 Test 1 sm-Tn5 0.25x 2 Test 2 "" "" 3 CH-P-A "" "" 4 CH-P-B "" "" 5 CH-P-C "" "" 6 CH-P-D "" "" 7 CH-O-A "" "" 8 CH-O-B "" "" 9 --(NTC) "" ""
Procedure[edit]
Prepare Enzyme Dilutions[edit]
- We want to dilute the supermutant-Tn5
- supermutant-Tn5 provided in 25x concentration, so dilute using the following:
0.5ulA (Tn5059 ME-A Tsm 25x) + 0.5ulB (Tn5059 ME-B Tsm 25x) + 99ul Tn5 standard storage buffer Tsm diluent
- We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 2ul ProtQ + 198ul Water
Protocol[edit]
- Prep tagmentation mastermix
1x 9.5x (ul) Template 1 -- Nxt 1 9.5 <- supermutant Tn5 HMW Buff 1 9.5 <- renamed "5x Tn5 Buffer" Water 2 19
- 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 9.5x (ul) 10U/ul Exo- 0.1 .95 25mM dNTP 0.4 3.8 Water 0.5 4.75
- 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 XT protocol recommendations for low plexity reactions found on File:2015 03 31 Nextera xt sample preparation guide.pdf. We also do not want to overlap adaptors from the previous libraries done on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-10>. Consequently, avoid using the combination of N701-705 and S505/506
rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) S502 (CTCTCTAT) 2 N702 (CGTACTAG) "" 3 N703 (AGGCAGAA) "" 4 N704 (TCCTGAGC) "" 5 N705 (GGACTCCT) "" 6 N706 (TAGGCATG) "" 7 N707 (CTCTCTAC) "" 8 N710 (CGAGGCTG) "" 9 (NTC) N711 ""
- 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[edit]
- Below is the gel image for this experiment (x,low mass ladder,x,rxn's1->9)
File:Cw ZhangLab 2 2015-03-31 16hr 42min catalina harbor sample sm-tn5059 lib prep.jpg
- From the gel image, it looks like library construction worked cleanly (NTC did not show up). Can continue doing sequencing when we have more samples to pool