Sam:LabNotes/Microbiome-new/2010-5-2

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Nextera (Epicentre) kit - Illumina sequencing library construction[edit]

Background[edit]

  • Test the library construction using Nextera kit
    • Dramatically shorten the time expense for library construction.
    • No need for DNA shearing.
    • Able for low-input DNA library construction (50ng).
    • Able for muplexing sequencing.

Sample preparation[edit]

  • Two E.coli MDA amplicons (made on 03-28-'10)were used for the current tests
  • Only use about 50ng DNA for starting amount
  • QUBIT-HS of DNA concentration:
    • 2hr-E.coli MDA amplicon: 1.17ng/uL in 50uL => use 50uL
    • 10hr-E.coli MDA amplicon: 819ng/uL in 50uL => diluted (1/100 ratio) into 8.19/uL => use 6.1 uL

Procedures[edit]

EtOH purification of MDA amplicons[edit]

    • Add 4.5uL tRNA carrier(100ng/uL) into MDA amplicon.
    • Add H2O to make total valume to 100uL.
    • Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 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.

Tagmentation reaction[edit]

  • Set up tagmentation reaction on cold block:
                          1rxn
DNA(50ng)+ H2O            15.0
5X Nextera Rxn buf.(LMW)   4.0
Nextera enzyme             1.0
------------------------------
                          20.0 uL
  • Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
  • During the incubation, setup the tube and buffer of Zymo DNA cleaning kit

Zymo DNA purification[edit]

  • Pre-load 100uL binding buf. in 1.5mL-tubes. Add 20uL reaction product from last step.
  • Briefly vortexing and centrifuging.
  • Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
  • Centrifuge at 10,000xg for 1min. Discard flowthrough.
  • Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flowthrough.
  • Repeat the last step again.
  • Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue).
  • Add 11uL pre-warmed H2O onto the column, transfer the column onto a 1.5mL-tube. Incubate at RT for 2min.
  • Centrifuge at 10,000xg for 1min. The elute DNA is about 10~11uL. Only use 5uL for the library amplification. (Label and store the other 5uL product in -20C)

Library enrichment by PCR amplification[edit]

  • Set up the reaction on cold block in PCR tubes:
                             1rxn
RT-PCR grade H2O             17.0
DNA lib (from last step)      5.0
2X Nextera PCR buf.          25.0
50X Nextera primier cocktail  1.0 - Illumina-compatible       
50X Nextera adaptor 2         1.0 - could be stwitch to barcoded library (optional)
Netera PCR enzyme             1.0
---------------------------------
                             50.0 uL
  • Briefly vortexing and centrifuging.
  • Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x12 cycle -> 4C forever
  • NOTE: the result should be about 500ng amplified DNA (Zymo manual).

Results[edit]

  • 3uL of amplified product was validated using TBE-PAGE
    • 3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
    • Run at 200V, 30min on a 10-well TBE-PAGE (Use Invitrogen premade gel).
    • Long staing (15mins) with foil cover.
    • Picture was taken at exposure time 4.096sec
File:Sam050210-nextera lib.jpg
Concentration:     2h-Ecoli MDA(50ng)   10h-Ecoli MDA(50ng)
QUBIT-HS (ng/uL)        17.0                 15.4
  • For using Low-molecular-weight(LMW) fragmentation buffer, the expeced size will be around 200-500bp. The result in gel picture support this point.
  • There still have some smear tail above 500bp. I am curious if it is caused by over PCR amplification.
    • Although the amplfication cycle (12 cycles) is suggested in protocol, I am inclined to test the result with 10cycles amplification using the remained 5uL templates.
    • A size selection may be necessary for removing large fragments.
  • NOTE: Condition summary
    • Starting material: 50ng MDA amplicon (non-sheared, but EtOH purified).
    • Using Zymo-DNA cleaning column
    • 12cycle amplification (5uL template in 50uL rxn volume).
File:Nextera kit - oversize of libraries.jpg