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

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Revision as of 19:59, 29 December 2010 by >Sam Chiang (→‎Sample preparation)
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Nextera-library construction on E.coli SAG(0706'10 MDA)

Background

Sample preparation

 Sample no.   Sample name          Conc.(ng/uL)  Sample(uL)      tRNA("2")   H2O    Total DNA(ng)
 ---------------------------------------------------------------------------------------------------
 NX01         WGA102710-(2-1)- 3hr     0.88         50.00           3.5       0.00    43.95ng
 NX02         WGA102710-(2-1)-10hr   729.07          0.68            -       49.00   500.00ng
 NX03         WGA102710-(2-4)- 3hr     0.98         50.00           3.5       0.00    48.95ng
 NX04         WGA102710-(2-4)-10hr   693.02          0.72            -       49.00   500.00ng
 
 *I didn't put any tRNA carrier for 4hr-amplified amplicons.
  • 50uL sample suspension were carefully transferred into microtubes.
  • Samples tubes were tapped on the rack and packed in a paper box with three disposable cooler.
  • The package was shipped to the Sara through FedEx 11-17-'10 by Alice.


  • Only use about 10ng DNA for starting amount (10ng is the total available amount for the sample Sam070610-90m-C)
    • Use all of remained 30uL for Sam070610-90m-C (0.3ng/uL, 50uL)=> (~10ng)
    • Make 1/100 dilution for Sam070610-90m-C-ext(379ng/uL, 20uL)=> 3.8ng/uL => Use 3uL of this diluted template (~10ng)
  • There is no need for doing DNA shearing because I am plan to use Nextera lib construction (Transposon-based)

EtOH purification of MDA amplicons

    • Add 4.9uL 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

  • 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

  • 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

  • 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)x10 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.