Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-10

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ETOH Precipitation w/ tRNA Carrier + Library Construction Test (Part II)[edit]

Background[edit]

rxn     Sample     Total Template Input     ETOH Precipitation?
1       UO2-NP     0.5ng                    Yes
2       UO3-NP     0.5ng                    Yes
--------------------------------------------------------------------
3       UO2-NP     0.5ng                    No
4       UO3-NP     0.5ng                    No
--------------------------------------------------------------------
5       UO2-NP     1ng                      No
6       UO3-NP     1ng                      No
--------------------------------------------------------------------
7       ETOH NTC   --                       Yes
8       NTC        --                       No

Protocol[edit]

Sample Dilution[edit]

  • 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
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

ETOH Precipitation w/ tRNA carrier[edit]

  • We will need to perform ETOH precipitation on the 38pg/ul diluted samples to simulate what we are going to do with the lower-concentrated samples. As a reminder, these lower-concentrated South Pacific Gyre samples will be in 11ul solution. Consequently, we need to prepare the following ETOH precipitation mastermix:
                            1x (ul)
Sample (38pg/ul)            11 <- Samples (total input of 0.5ng): UO2-NP, UO3-NP, H2O (RXN 1 & 2 & 7)
tRNA Carrier (100ng/ul)     2
Water                       8
100% ETOH                   50
3M NaOAc                    2
Glycoblue                   1.5
  • Perform same protocol as general protocol but elute in 3ul Water + 1ul HMW Buffer (Note: This may be difficult and may need to be adjusted if this experiment does not work)
  • We also want to make sure that the change in total input DNA (to ~0.5ng) will not affect the reaction. Consequently, we will make another set of reactions in the following mastermix:
                    1x (ul)
Sample (1ng/ul)     0.5 <- Samples (total input of 0.5ng): UO2-NP, UO3-NP (RXN 3 & 4)
HMW Buff            1
Water               2.5
  • At the same time, also mix the control reactions (with 1ng/ul template) with HMW Buffer in the following mastermix:
                    1x (ul)
Sample (1ng/ul)     1 <- Samples (total input of 1ng): UO2-NP, UO3-NP, H2O (RXN 5 & 6 & 8)
HMW Buff            1
Water               2

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]

  • Add the Nextera enzyme to each of the already mixed template/HMW/water solutions
                       1x (ul)
Template/HMW/Water     4 (already prepared previously)
Nxt                    1 <- supermutant Tn5
  • 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      9x(ul)
10U/ul Exo-     0.1     .9
25mM dNTP       0.4     3.6
Water           0.5     4
  • 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 3/10 or 3/31. Consequently, avoid using the combination of N701-705 and S505/506/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         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