Sam:LabNotes/Microbiome-new/2010-8-4: Difference between revisions

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===Library enrichment by PCR amplification===
===Library enrichment by PCR amplification===
*Set up the reaction on cold block in PCR tubes:
*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
                               1rxn
  RT-PCR grade H2O            17.0
  RT-PCR grade H2O            17.1
  DNA lib (from last step)      5.0
  DNA lib (from last step)      5.0
  2X Nextera PCR buf.          25.0
  2X Phusion master mix        25.0
  50X Nextera primier cocktail  1.0 - Illumina-compatible       
  50X Nextera primier cocktail  1.0 - Illumina-compatible       
  50X Nextera adaptor 2        1.0 - could be stwitch to barcoded library (optional)
  50X Nextera adaptor 2        1.0 - Using IDX1 and IDX2
  Netera PCR enzyme            1.0
  50X SYBR I                    0.4
Isothermo(Epicentre)          0.5
  ---------------------------------
  ---------------------------------
                               50.0 uL
                               50.0 uL
*Briefly vortexing and centrifuging.
*Briefly vortexing and centrifuging.
*Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x10 cycle -> 4C forever
 
 
 
 
 
**65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15C -> 72C 20sec)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).
*'''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).



Revision as of 21:45, 6 August 2010

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

Background

  • 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

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

    • 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

  • 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 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

  • 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 15C -> 72C 20sec)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 WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.

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).