Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31: Difference between revisions

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>Cjwei
(Created page with "=Nextera Lib Construction on Catalina Harbor Samples= ==Background== *Previously, I tested the supermutant Tn5 on samples CH-S-A/B/C/D and found that the 0.25x concentration w...")
 
>Cjwei
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*We want to dilute the supermutant-Tn5
*We want to dilute the supermutant-Tn5
**supermutant-Tn5 provided in 25x concentration, so dilute using the following:
**supermutant-Tn5 provided in 25x concentration, so dilute using the following:
  0.5ulA + 0.5ulB + 99ul dilution buffer
  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
*We also want to dilute ProtQ 1/100 from 20mg/ml.  In order to do this, we add 2ul ProtQ + 198ul Water
===Protocol===
===Protocol===
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  Template    1      --
  Template    1      --
  Nxt          1      9.5 <- supermutant Tn5
  Nxt          1      9.5 <- supermutant Tn5
  HMW Buff    1      9.5
  HMW Buff    1      9.5 <- renamed "5x Tn5 Buffer"
  Water        2      19
  Water        2      19
*Incubate: 55C 5min -> remove tubes and put on ice block
*Incubate: 55C 5min -> remove tubes and put on ice block
Line 51: Line 51:
  Water          0.5    4.75
  Water          0.5    4.75
*Incubate: 37C 15min -> 75C 20min -> 10C forever
*Incubate: 37C 15min -> 75C 20min -> 10C forever
*Add the following i5 adaptor (N/S5xx, containing index 2) and 1ul i7 adaptor (N7xx, containing index 1) in the following order.  This is based off of the Nextera protocol recommendations for low plexity reactions found on [[File:cw_2015_02_26_Nextera_DNA_Sample_Prep_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>.  Consequently, avoid using the combination of N701-705 and N505/506'''
*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 <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-10>.  Consequently, avoid using the combination of N701-705 and S505/506'''
  rxn #    Indx 1 (i7)        Indx 2 (i5)
  rxn #    Indx 1 (i7)        Indx 2 (i5)
  1        N701 (TAAGGCGA)    S502 (CTCTCTAT)
  1        N701 (TAAGGCGA)    S502 (CTCTCTAT)
Line 61: Line 61:
  7        N707 (CTCTCTAC)    ""
  7        N707 (CTCTCTAC)    ""
  8        N710 (CGAGGCTG)    ""
  8        N710 (CGAGGCTG)    ""
9 (NTC)  N711                ""
   
   
*Prep PCR mastermix -> add 11ul to each rxn
*Prep PCR mastermix -> add 11ul to each rxn
Line 70: Line 71:
*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 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|500px]]
*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

Latest revision as of 18:45, 1 April 2015

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