Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-22

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Adjusted Tn5 Rxn Volume Test[edit]

Background[edit]

  • Previously, I tried ETOH precipitation with tRNA carriers in order to concentrate the sample to fit into the 5ul Tn5 reaction (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-10>). However, that experiment failed.
  • In order to get around ETOH precipitation, we will try to do another test in which we increase the volume of the Tn5 reaction to account for the most dilute samples (38pg/ul in 11ul). In order to do this, we will increase the reaction volume to 15ul (instead of 5ul) and use only 10ul of original sample
  • Below is a summary of the reactions for this experiment:
rxn     Sample     Template Input     Enzyme Vol (0.25x conc)
1       UO2-NP     10ul 38pg/ul       1ul
2       UO3-NP     10ul 38pg/ul       1ul
-------------------------------------------------------------
3       UO2-NP     10ul 38pg/ul       2ul
4       UO3-NP     10ul 38pg/ul       2ul
-------------------------------------------------------------
5       UO2-NP     1ul 1ng/ul         1ul
6       UO3-NP     1ul 1ng/ul         1ul
-------------------------------------------------------------
7       UO2-NP     1ul 1ng/ul         2ul
8       UO3-NP     1ul 1ng/ul         2ul
-------------------------------------------------------------
9       NTC        --                 1ul

Procedure[edit]

Sample Dilution[edit]

  • We want to dilute the samples to 1ng/ul using the following dilutions (this has already been done in the previous ETOH test). 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
UO2-NP     1.81ng/ul         2             1.62
UO3-NP     3.67              1             2.67
  • 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
UO2-NP     1ng/ul            1             25.32
UO3-NP     1                 1             25.32

Prepare Enzyme Dilutions[edit]

  • 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 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 reactions (do them all separately and add enzyme last)
rxn     Sample     Template         5x Tn5 Buff     Water     Enzyme Vol (0.25x conc)
1       UO2-NP     10ul 38pg/ul     3ul             1ul       1ul
2       UO3-NP     10ul 38pg/ul     3               1         1
-------------------------------------------------------------
3       UO2-NP     10ul 38pg/ul     3               --        2
4       UO3-NP     10ul 38pg/ul     3               --        2
-------------------------------------------------------------
5       UO2-NP     1ul 1ng/ul       3               10        1
6       UO3-NP     1ul 1ng/ul       3               10        1
-------------------------------------------------------------
7       UO2-NP     1ul 1ng/ul       3               9         2
8       UO3-NP     1ul 1ng/ul       3               9         2
-------------------------------------------------------------
9       NTC        --               3               11        1
  • 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      9.5x (ul)
10U/ul Exo-     0.1     0.95
25mM dNTP       0.4     3.8
Water           0.5     4.75
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                ""

  • 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

Results[edit]

  • Below is the gel image from the experiment (Low mass ladder, rxn's 1->9, x, Dinh's lane [ignore])

File:Cw ZhangLab 2 2015-04-22 17hr 23min nxt volume test.jpg