Sam:LabNotes/Microbiome-new/2010-12-29: Difference between revisions

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>Sam Chiang
>Sam Chiang
Line 48: Line 48:
*Elute DNA in 11uL pre-warmed EB buffer.
*Elute DNA in 11uL pre-warmed EB buffer.


===Library enrichment by PCR amplification===
===Bridge-PCR(bPCR) amplification===
*Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
*Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
                               1rxn    4.5rxn
                               1rxn    4.5rxn

Revision as of 06:42, 30 December 2010

Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10)

Background

Sample preparation

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

  • 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

  • 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 were purified using MiniElute column
    • 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

  • 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     4.5 - Using barcoded adaptor (ID1 - ID4)
Nextera PCR enzyme            1.0     4.5
20X Eva-green                 1.0     4.5
-----------------------------------------
                             50.0   202.5 uL /4.5= 45 --- 5uL template
*Eva-green or SYBR-green acceptable range is from 0.2X~0.4X in rt-PCR reaction
  • Briefly vortexing and centrifuging.
  • Pre-cut the tubes
  • 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).

Ampure bead purification

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

Results

  • 3uL of amplified product was validated using TBE-PAGE
    • 3uL PCR product + 3uL 0.5X TBE + 3uL 6X loading dye.
File:Samxxxxxx-Nextera lib.jpg