Sam:LabNotes/Microbiome-new/2010-5-2
Jump to navigation
Jump to search
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
- Set up tagmentation reaction on cold block:
1rxn DNA(50ng)+ 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. (Store another 5uL product in -20C)