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       CH-S-A     1ul 1ng/ul         2ul 0.25x <- Verify results from previous test (rxn's 7/8 on 4/22)
8       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       CH-S-A     1ul 1ng/ul         3               9         2 (0.25x) <- Verify results from previous test (rxn's 7/8 on 4/22)
8       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      8.5x (ul)
10U/ul Exo-     0.1     0.85
25mM dNTP       0.4     3.4
Water           0.5     4.25
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         N707 (CTCTCTAC)     ""
8 (NTC)   N710                ""
  • 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