Sam:LabNotes/Microbiome-new/2010-12-29

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Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10) - test 1[edit]

Background[edit]

Sample preparation[edit]

Trial-1(12-29-'10)

 Sample no.   Sample name          Conc.(ng/uL)  Sample(uL)      tRNA("2")   H2O    Total DNA(ng)
 ---------------------------------------------------------------------------------------------------
 NX01         WGA102710-(1-4)- 3hr     5.77         50.00           0.0       0.00   288.50ng
 NX02         WGA102710-(1-4)-10hr  1162.79          0.43            -       49.00   500.00ng
 NX03         WGA110310-(5)-   3hr     2.29         50.00           0.0       0.00   114.50ng
 NX04         WGA110310-(5)-  10hr   555.81          0.90            -       49.00   500.00ng
  • I am trying not to added any tRNA carrier in for EtOH precipitation since the samples amount for short-amplification is around 100~300ng.

EtOH purification of MDA amplicons[edit]

  • Total volume: 50uL.
    • UV-decontamination the 100% EtOH, 3M NAOAc and GlycoBlue in crosslinker oven for 20min.
    • Add 2.5X volume 100% EtOH (125uL) + 0.1X volume 3M NaOAc (5uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
    • Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH.
    • Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O.
  • NOTE: White big pellets still showed in short-amplified samples which suggested their appearance has nothing to do with over-dose tRNA adding or the introduced contamination during Covaris shearing (in HMS by Sara). The white pellet may due to MDA regent content, either N6 primers or enzyme proteins.

Tagmentation reaction[edit]

  • Set up tagmentation reaction on cold block:
                          1rxn   4.5rxn
DNA template+H2O          15.0     -
5X Nextera Rxn buf.(LMW)   4.0   18.0
Nextera enzyme             1.0    4.5
-------------------------------------
                          20.0   22.5/4.5 = 5uL -- template 15uL
  • Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
  • During the incubation, setup the tube and buffer of MiniElute columns.
  • Reaction products (20uL) were purified using MiniElute column following the standard QIAquick protocol.
    • MiniElute columns have lids which are better to avoid introduced contamination.
    • The efficiency of MiniElute columns has been verified in Nextera-kit manual.
  • Elute DNA in 11uL pre-warmed EB buffer.

Bridge-PCR(bPCR) amplification[edit]

  • Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
                             1rxn    4.5rxn
RT-PCR grade H2O             16.0    72.0
DNA lib (from last step)      5.0     -
2X Nextera PCR buf.          25.0   112.5 
50X Nextera primier cocktail  1.0     4.5 - Illumina-compatible       
50X Nextera adaptor 2         1.0     -   - Using barcoded adaptor (ID1 - ID4)
Nextera PCR enzyme            1.0     4.5
20X Eva-green                 1.0     4.5
-----------------------------------------
                             50.0   198.0 uL /4.5= 44 --- 5uL template + 1uL barcoded adaptor
*Eva-green or SYBR-green acceptable range is from 0.2X~0.4X in rt-PCR reaction
  • Briefly vortexing and centrifuging.
  • Cut the tubes from the strip.
  • Program "Nextera lib amplification with Nextera-PCR enzyme": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x15 cycle -> 4C forever
    • Pick up sample around 9 cycles (for 50ng-template reactions).

Results[edit]

  • Samples were picked from Realplex machine when they arrived about 10000~15000 increasement of fluorescence.
File:Sam122910-qPCR-1.jpg Monitored fluorescence
File:Sam122910-qPCR-3.jpg Normalized fluorescence
 Sample      NX1    NX2   NX3   NX4
             ----------------------
 Well        A1           A3       
                    B2          B4 
 Min         33     29    58    29 
 Cycle        7      6    13     6 
 Dif. Fluorescence(monitored) 
          12000   8000 11000  8000  
  
  • The starting fluorescent intensities for each of templates are consistent with their estimated DNA concentration (by QUBIT-HS), which suggested that the QUBIT-HS quantification is relatively accurate.
Library    DNA starting amount    qPCR starting fluorescence(monitored)
NX1        288ng                  10000 
NX2        500ng                  17000
NX3        114ng                   4000
NX4        500ng                  16000

Libraries validation / quantification[edit]

  • Amplified libraries were validated by TBE-PAGE. Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2
    • 3uL PCR products were run on 10-well PAGE (200V, 30min).
    • Sample well: 1uL sample + 3uL 6X loading dye + 6uL 0.5X TBE buffer
    • Ladder well: 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
      • Load 6uL ladder on the 1st well and 3uL on the 2nd well
    • Use 3uL SYBR-gold with regular staining(10min) with foil protection
File:Sam122910 lib-3.jpg  File:Sam122910 lib-quant-2.jpg
File:Sam122910-regression line.jpg
                 Total yield(ng)
NX1 2.51 ng/uL   125.5 ng
NX2 1.27 ng/uL    63.5 ng
NX3 0.67 ng/uL    33.5 ng
NX4 0.30 ng/uL    15.0 ng

Discussion[edit]

  • Nextera kit can work up to 500ng template.
  • Nextera kit works better with 50-100ng template. Too much tempalte (300ng-500ng) will cause the problem on tagmentation reaction(using LMW buffer). The library DNA will not be distributed with 200~400bp properly.
  • It is unclear why library NX3 needs to be amplified to the 13th cycle. However it showed the best size distribution within 200~400bp.