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

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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.

Library enrichment by PCR amplification

  • Set up the reaction on cold block in PCR tubes:
                             1rxn    4.5rxn
RT-PCR grade H2O             17.0    76.5
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)
Netera PCR enzyme             1.0     4.5
-----------------------------------------
                             50.0   202.5 uL /4.5= 45 --- 5uL template
  • Briefly vortexing and centrifuging.
  • Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x15 cycle -> 4C forever
  • NOTE: The manual suggested only amplify 9 cycle. Since my starting amount is only 10ng (instead of 50ng), I amplify one more cycle (total 10cycle).

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:Sam072410-Nextera lib.jpg
  • I accidently over-run the gel (250V, 30min). I should only use 200V instead.
  • The bands showed up but look smear.
    • For the 10ng starting amount, 10cycle PCR amplification is not enough.

Extra-library amplification using rt-PCR

  • Since there are 10uL template left from the 1st step of Nextera library construction. Only 5uL were used for PCR amplification.
  • I am trying to use the other 5uL template(transposon tagged DNA) for amplification.
  • Set up the reaction on cold block in EP-white PCR-strip tubes:
                             1rxn     x2.2 rxn
RT-PCR grade H2O             16.8     36.96
DNA lib (from last step)      5.0       -
2X Nextera PCR buf.          25.0     55.00
50X Nextera primier cocktail  1.0      2.20  - Illumina-compatible       
50X Nextera adaptor 2         1.0      2.20  - could be stwitch to barcoded library (optional)
Netera PCR enzyme             1.0      2.20  
50X SYBR-green I              0.2      0.44
-------------------------------------------
                             50.0     99.00/2.2=45 --- template 5uL
NOTE: I use the "no.2" SYBR-green 50X
Stop the reaction before the amplification curve reach pleatue

Results

  • 3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(200V, 30min).
File:Sam072910-nextera lib amp-2nd amp.jpg
  • The 90min-library showed good range between 200-400bp
  • The 10hr-library is probably over-loaded on gel. I need to do the nanodrop on this amplicon and validate again.
    • After AMpure amplification the concentration could be raised up becasued the totoal volume was changed from 50uL to 30uL (I used 30uL H2O to elute the purified DNA).
  • The comparison of before and after AMpure bead purification showed no significatn loss with the purification method.