Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-23: Difference between revisions

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  5      CH-S-A    10ul 38pg/ul      0.25x * 1/16d
  5      CH-S-A    10ul 38pg/ul      0.25x * 1/16d
  6      CH-S-A    1ul 1ng/ul        0.25x <- Pos Control
  6      CH-S-A    1ul 1ng/ul        0.25x <- Pos Control
  7      NTC        --                0.25x <- NTC
  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==
==Procedure==
===Sample Dilution===
===Sample Dilution===
Line 40: Line 41:
  5      CH-S-A    10ul 38pg/ul      3              1        1 (0.25x * 1/16d)
  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
  6      CH-S-A    1ul 1ng/ul        3              10        1 (0.25x) <- Pos Control
  7      NTC        --                3              11        1 (0.25x) <- NTC
  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
*Incubate: 55C 5min -> remove tubes and put on ice block
*Add 1ul 1/100d ProtQ to each reaction
*Add 1ul 1/100d ProtQ to each reaction
*Incubate: 50C 10min -> 70C 20min -> 10C forever
*Incubate: 50C 10min -> 70C 20min -> 10C forever
*Prep Exo- mastermix (add 1ul to each rxn)
*Prep Exo- mastermix (add 1ul to each rxn)
                 1x      8x (ul)
                 1x      8.5x (ul)
  10U/ul Exo-    0.1    0.8
  10U/ul Exo-    0.1    0.85
  25mM dNTP      0.4    3.2
  25mM dNTP      0.4    3.4
  Water          0.5    4.0
  Water          0.5    4.25
*Incubate: 37C 15min -> 75C 20min -> 10C forever
*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'''
*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'''
Line 58: Line 60:
  5        N705 (GGACTCCT)    ""
  5        N705 (GGACTCCT)    ""
  6        N706 (TAGGCATG)    ""
  6        N706 (TAGGCATG)    ""
  7 (NTC)  N707 (CTCTCTAC)    ""
  7         N707 (CTCTCTAC)    ""
8 (NTC)  N710                ""
*Add 21ul KAPA SYBR Fast (2x) to each rxn
*Add 21ul KAPA SYBR Fast (2x) to each rxn
*Incubate: 72C 3min -> [98C 10sec -> 63C 30sec -> 72C 3min]x13 -> 10C
*Incubate: 72C 3min -> [98C 10sec -> 63C 30sec -> 72C 3min]x13 -> 10C
Line 64: Line 67:
*Run 3ul on TBE gel
*Run 3ul on TBE gel
*Purify again (same ratio) before sequencing if low-weight fragments appear on gel
*Purify again (same ratio) before sequencing if low-weight fragments appear on gel
==Results==
*Below is the gel image from this experiment (x, Low Mass Ladder, x, rxn's 1->8)
[[File:cw_ZhangLab_2_2015-04-23_21hr_11min_ntx_volume_test_2.jpg|500px]]

Latest revision as of 20:41, 24 April 2015

Adjusted Tn5 Rxn Volume Test II[edit]

Background[edit]

  • 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[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
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[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 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[edit]

  • 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

Results[edit]

  • Below is the gel image from this experiment (x, Low Mass Ladder, x, rxn's 1->8)

File:Cw ZhangLab 2 2015-04-23 21hr 11min ntx volume test 2.jpg