Sam:LabNotes/Microbiome-new/2010-11-07 exp1

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Illumina library construction - E.coli SAG samples - 3rd test[edit]

Background[edit]

1. No 2nd MDA for short-amplified amplicons to avoid the over-dose N6 primer.
2. In order to reduce the possible effect from tRNA, I reduced the amount of added tRNA carrier. For short-amplified samples (40~50ng DNA) only 350ng tRNA carrier were added.
3. Samples are diluted in 50uL(total volume) for shearing. Since 100uL sample is very full for Covaris microtubes (50uL in microtubes works fine in previous experiments).

Procedures[edit]

DNA purification[edit]

  • Transfer 50uL sheared samples into 1.5mL non-sticky tubes(Ambion). Add 50uL RT-PCR grade H2O to make total volume into 100uL.
    • No additional tRNA-carrier was added to any sample.
  • Add 250uL 100% EtOH(2.5V) + 10uL 3M NaOAc(0.1V) + 1.5uL Glycoblue. Invert 20times. Incubate at -80C for 25min.
  • Centrifuge at 10,000xg at 4C for 25min. Remove supernatant. Wash the pellet with 750uL cold 75% EtOH. NOTE: I saw relatively smaller white pellets for short-amplified samples. No pellet-breaking during the 75% EtOH washing.
  • Centrifuge at 10,000xg at 4C for 5min. Remove the supernatant. Air dry the pellet for 5min. Elute the purified DNA pellet in 34uL RT-PCR grade H2O. Store the DNA in -20C

End-repairing[edit]

  • End-it Kit (EPICENTRE)
    • Prepare master-mix in 0.5-mL tube on cold block.
    • Set up reaction in individual PCR tubes on cold block.
                      1rxn     4.5rxn
DNA+H2O               34.0      -
10X buf.               5.0     22.5
2.5mM dNTP             5.0     22.5
10mM  ATP              5.0     22.5
End-it enzyme          1.0      4.5
------------------------------------
                      50.0     72.0/4.5=16 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
  • Purify the DNA with QIAquick. Elute DNA with 28uL pre-warmed EB (14uL x2, 1min incubation at RT)

A-tailing[edit]

  • Exo-minus klenow DNA pol. kit (Epicentre)
    • Prepare master-mix in 0.5-mL tube on cold block.
    • Set up reaction in individual PCR tubes on cold block.
                       1rxn     4.5rxn
DNA+H2O                28.0      -
10X buf.                4.0     18.0
1mM dATP                6.0     27.0
Exo-minus klenow enzyme 2.0      9.0
------------------------------------
                       40.0     54.0/4.5=12 
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
  • QIAquick purification. Eluted DNA with pre-warmed 12uL EB(2min incubation at RT).

Ligation[edit]

  • T4 ligation kit (Enzymatic)
    • Prepare master-mix in 0.5-mL tube on cold block.
    • Set up reaction in individual PCR tubes on cold block.
                      1rxn      4.5rxn
DNA+H2O               11.0       -
2X rapid buf.         15.0     67.5
20uM Y-adaptor         2.0      9.0 
T4 ligase enzyme       2.0      9.0
------------------------------------
                      30.0     85.5/4.5=19 
  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • QIAquick purification. Eluted DNA with pre-warmed 20uL EB (10uL x2, 1min incubation at RT).

Size selection[edit]

  • 20uL PCR product samples were run on 5-well PAGE (200V, 30min).
    • Sample well: 20uL sample + 10uL 6X loading dye + 10uL RT-PCR grade H2O
    • Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL RT-PCR grade H2O
File:Sam110810-lib.jpg  File:Sam110810-lib postcut.jpg
Ligation product                                    Ligation product (post gel-cut)


New ID            HC110910-1  HC110910-2  HC110910-3  HC110910-4  HC110910-5  HC110910-6  HC110910-7  HC110910-8
----------------------------------------------------------------------------------------------------------------
Original ID       SAM1        SAM2        SAM3        SAM4        1S          2S          1L          2L
Original DATE     110610      110610      110610      110610      102510      102510      102510      102510
Lib Construction 
  • For all of short-amplified samples I add 4.5uL tRNA(96ng/uL) to make the total nucleotide to 500ng before DNA recovery from gel-slice.
  • Shred the gel slice using pole-punctured 0.5mL non-sticky tubes. Centrifuge at 14,000rpm for 3min.
  • Incubate the gel slice mixture with 500uL clean 1X TE at 37C for 2hr on the vortexer.
    • NOTE: I did a over-night incubation for this step.
  • Pellet the gel suspension at 14,000rpm for 1.5min at RT.
  • Transfer the supernatant onto Nanosap filter. Centrifuge at 14,000rpm for 1.5min at RT.
    • NOTE: Usually the large gel slice get fewer supernatant. I got 350uL for the long-amplified samples and 300uL for the short-amplified samples.
  • Transfer the filtrate into clean 1.5-mL non-sticky tubes.
  • EtOH precipitation: 2.5V EtOH, 0.1V 3M NaOAc, 1.5uL Glycoblue. Mix well by inverting and incubate at -80C for 25min or more.
  • Centrifuge at 10,000xg 4C for 25min. Wash pellet with 75% EtOH, centrifuge at 10,000xg 4C for 5min. Air dry 5min. Elute the pellet in 20uL H2O.

PCR amplification (ligation PCR) with N2(kz)index primer[edit]

 Lib-amp PCR(100uL): Dr.Zhang recipe

                             1rxn   8.5 rxn
 H2O                         34     289  μl
 Adapter ligated DNA         10       -  μl
 PCR_F(10uM)                  2      17  μl
 PCR_R.N2 Index(10uM)         2       -  μl
 20X EVA Green                2      17  μl
 2X Phusion HF MM            50     425  μl
                           ----------------
                            100 μl  748/8.5=88 --- template 10uL, Index primer(R) 2uL

  • The reactions tubes were pre-cut before incubation.
  • Using new two-step PCR program. qPCR was performed using Eppendorf realplex machine:
    • 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles -> (98C 10sec -> 72C 20sec) x 10 cycles -> 72C 3min -> hold 15C.

Results[edit]

  • Samples were picked from Realplex machine when they arrived about 10000~15000 increasment (monitored) of fluorescence.
File:Sam110910-qPCR-1.jpg Monitored fluorescence
File:Sam110910-qPCR-2.jpg Normalized fluorescence
             1      2     3     4     5     6     7     8
             ---------------------------------------------
             A1           A3          A5          A7
                    B2          B4          B6          B8
 Min         17     17    11    11    18    18    12    13
 Cycle       13     13     6     6    15    18     7     8
 Dif. Fluorescence(monitored) 
          11000  11000 16000 16000 15000 16000 17000 13000
 
  • 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: 3uL sample + 3uL 6X loading dye + 3uL 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 stainig(5min) with foil protection
File:Sam110910-amp lib new.jpg

Follow up (11/11/'10)[edit]

  • I will quantify the amplified library and pool them together for another size selection to get a narrow region for sequencing.
  • The results above are only came from half of ligation products(10uL). I decided to amplify another half of ligation products to get higher total product for 2nd size selection.
File:Sam111110-amp lib new.jpg
  • By using the same setting for amplification, I almost got the exactly same result as the previous amplification.