Sam:LabNotes/Microbiome-new/2010-8-4

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Nextera-library construction on single E.coli MDA amplicons(0727'10 MDA)[edit]

Background[edit]

  • The positive amplicons are:
    • Well-3(E)-10hr-sam072710, well-4(F)-10hr-sam072710
    • I did the QUBIT-HS for both amplcons and their original 90m amplicons.
File:Sam080410 - QUBIT -new.jpg
  • Four samples will be made Nextera libraries with barcoded primers.

Sample preparation[edit]

Name    DNA(20ng)   tRNA carrier(480ng)   RT-PCR H2O
----------------------------------------------------
90m-3   45.8        4.8                   49.4
10h-3    2.0        4.8                   93.2
90m-4   40.0        4.8                   55.2
10h-4    2.3        4.8                   92.9
  • Only use about 20ng DNA for starting amount (10ng is the total available amount for the sample Sam072710-90m-3)


EtOH purification of MDA amplicons[edit]

  • Add 4.8uL 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(10ng)+ 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 flow-through.
  • 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]

  • In order to use the libraries for cloning sequencing, we have to switch to the Phusion enzyme instead.
    • The Phusion enzyme provide blunt end PCR products which is suitable for blunting cloning.
    • We also need to add Isotherm (Epicentre) enzyme to repair the gaps left by transposon taggmentation.
  • In order to test the new amplification recipe, I only tried two of four samples with barcoded(Index) primer
    • 90m-4(IDX1) and 10hr-4(IDX2)
  • Set up the qPCR reaction (50uL) on cold block in PCR tubes:
                             1rxn
RT-PCR grade H2O             17.1
DNA lib (from last step)      5.0
2X Phusion master mix        25.0
50X Nextera primier cocktail  1.0 - Illumina-compatible       
50X Nextera adaptor 2         1.0 - Using IDX1 and IDX2 
50X SYBR I                    0.4
Isothermo(Epicentre)          0.5
---------------------------------
                             50.0 uL
  • Briefly vortexing and centrifuging.
    • 65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15sec -> 72C 20sec)x15 cycle -> 72C 3min -> 4C forever
    • NOTE1.: This program is adapted from Dr.Zhang's protocol 0805'10. The temperature setting is different from original Nextera protocol.
    • NOTE2.: The 65C 3min is for Isothermo enzyme. The 98C 30sec is for Phusion (Hot-start) enzyme.
  • Stop the reaction before the amplification reach saturation.
    • Sample 90m-4 stopped at 16cycle
    • Sample 10h-4 stopped at 8cycle

Ampure bead purification[edit]

  • Following standard protocol with 0.7V volume AMpure beads.
  • Nanodrop the purified DNA(optional) and stored it in -20C immediately.

Results[edit]

  • 3uL of amplified product was validated using TBE-PAGE
  • 3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(250V, 20min).
    • 3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
File:Sam080510- Nextera lib amp - phusion EZ.jpg File:Sam080610- Nextera-lib amplified.jpg
  • The templates from these qPCR amplification is 10ng
    • Start with 20ng but only 1/2 of 1-step product were amplified.
    • The sample 10h-4 may contain more template than 90m-4 so it was amplified faster.
    • The sample 10h-4 was prepared by dilution method to get estimated 20ng as starting amount. It is possible that the inaccuracy of quantification cause higher template used in 10h-4 sample.
  • Using 10ng templates for qPCR is not stable in this method, even with 16cycle amplification the PCR amplicons still hard to be seen. However if I keep amplifying the background may start showing up.