Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31

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Nextera Lib Construction on Catalina Harbor Samples[edit]

Background[edit]

                      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
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