Sam:LabNotes/Microbiome-new/2010-5-2: Difference between revisions

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>Sam Chiang
(Created page with '='''Nextera (Epicentre) kit - Illumina sequencing library construction''''= ==Background== *Test the library construction using Nextera kit **Dramatically shorten the time expe…')
 
>Sam Chiang
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**Able for low-input DNA library construction (50ng).
**Able for low-input DNA library construction (50ng).
**Able for muplexing sequencing.
**Able for muplexing sequencing.
==Sample preparation==
*Two E.coli MDA amplicons (made on 03-28-'10)were used for the current tests
*Only use about 50ng DNA for starting amount
*QBIT-HS of DNA concentration:
**2hr-E.coli  MDA amplicon: 1.17ng/uL in 50uL => use 50uL
**10hr-E.coli MDA amplicon: 819ng/uL  in 50uL => diluted (1/100 ratio) into 8.19/uL => use 6.1 uL
==Procedures==
===EtOH purification of MDA amplicons===
**Add 4.5uL 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===
*

Revision as of 09:02, 3 May 2010

Nextera (Epicentre) kit - Illumina sequencing library construction'

Background

  • Test the library construction using Nextera kit
    • Dramatically shorten the time expense for library construction.
    • No need for DNA shearing.
    • Able for low-input DNA library construction (50ng).
    • Able for muplexing sequencing.

Sample preparation

  • Two E.coli MDA amplicons (made on 03-28-'10)were used for the current tests
  • Only use about 50ng DNA for starting amount
  • QBIT-HS of DNA concentration:
    • 2hr-E.coli MDA amplicon: 1.17ng/uL in 50uL => use 50uL
    • 10hr-E.coli MDA amplicon: 819ng/uL in 50uL => diluted (1/100 ratio) into 8.19/uL => use 6.1 uL

Procedures

EtOH purification of MDA amplicons

    • Add 4.5uL 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