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 No edit summary |
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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.