Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-23
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Adjusted Tn5 Rxn Volume Test II
Background
- Yesterday, I tried adjusting the Tn5 reaction volume to 15ul (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-22>). However, only reactions 5&6 showed good smears (with rxn's 7&8 being very dim). This means that the 1ul 0.25x supermutant Tn5059 was sufficient for digesting 1ng of DNA but when we increased that to 2ul 0.25x Tn5059, there was over-digestion. Consequently, it looks like for the ~400pg DNA template input, I likewise over-digested the template leading to no product on the gel.
- Today, I'll be working on another test, this time trying out different enzyme concentration inputs in order to lower the concentration to get some product.
- In order to not run out of template, I'll be reverting back to the Catalina Harbor samples (particularly testing with CH-S-A, which was 63.2ng/ul). Consequently, I'll be running the following test:
rxn Sample Template Input Enzyme Conc (1ul added) 1 CH-S-A 10ul 38pg/ul 0.25x 2 CH-S-A 10ul 38pg/ul 0.25x * 1/2d 3 CH-S-A 10ul 38pg/ul 0.25x * 1/4d 4 CH-S-A 10ul 38pg/ul 0.25x * 1/8d 5 CH-S-A 10ul 38pg/ul 0.25x * 1/16d 6 CH-S-A 1ul 1ng/ul 0.25x <- Pos Control 7 NTC -- 0.25x <- NTC
Procedure
Sample Dilution
- We want to dilute the samples to 1ng/ul using the following dilutions. These will be utilized for our positive control when performing library construction on the supermutant (we will be utilizing exactly 1ul of the 1ng/ul sample for the positive control)
Concentration ul Sample ul Water CH-S-A 63.2ng/ul 1ul 62.2ul
- In order to make the concentration more representative of the lower concentrated South Pacific Gyre samples, we will further dilute to 38pg/ul (0.038ng/ul) using the following dilutions: (Note: We are utilizing the 1ng/ul dilution fo the sample and further diluting it down to 38pg/ul)
Concentration ul Sample ul Water CH-S-A 1ng/ul 1 25.32
Prepare Enzyme Dilutions
- We want to dilute the supermutant-Tn5 (This was already done previously on the 3/31/2015 library construction experiment and we will use the same diluted enzymes)
- 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 want to further dilute the enzyme from 0.25x with the following serial dilution:
Starting Conc ul Tn5 ul Water Ending Conc 0.25x 2 2 0.25x * 1/2d 0.25x * 1/2d 2 2 0.25x * 1/4d 0.25x * 1/4d 2 2 0.25x * 1/8d 0.25x * 1/8d 2 2 0.25x * 1/16d
- We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 2ul ProtQ + 198ul Water
Protocol
- Prep tagmentation reactions (do them all separately and add enzyme last)
rxn Sample Template Input 5x Tn5 Buff Water Enzyme Vol (Conc) 1 CH-S-A 10ul 38pg/ul 3ul 1ul 1ul (0.25x) 2 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/2d) 3 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/4d) 4 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/8d) 5 CH-S-A 10ul 38pg/ul 3 1 1 (0.25x * 1/16d) 6 CH-S-A 1ul 1ng/ul 3 10 1 (0.25x) <- Pos Control 7 NTC -- 3 11 1 (0.25x) <- NTC
- Incubate: 55C 5min -> remove tubes and put on ice block
- Add 1ul 1/100d ProtQ to each reaction
- Incubate: 50C 10min -> 70C 20min -> 10C forever
- Prep Exo- mastermix (add 1ul to each rxn)
1x 8x (ul) 10U/ul Exo- 0.1 0.8 25mM dNTP 0.4 3.2 Water 0.5 4.0
- Incubate: 37C 15min -> 75C 20min -> 10C forever
- Add 2ul i5 adaptor (S5xx, containing index 2) and 2ul i7 adaptor (S7xx, 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> or the successful libraries on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-22>. Consequently, avoid using the combination of N701-705 & S505/506, N705/706 & S502
rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) S503 (TATCCTCT) 2 N702 (CGTACTAG) "" 3 N703 (AGGCAGAA) "" 4 N704 (TCCTGAGC) "" 5 N705 (GGACTCCT) "" 6 N706 (TAGGCATG) "" 7 (NTC) N707 (CTCTCTAC) ""
- Add 21ul KAPA SYBR Fast (2x) to each rxn
- 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