Sam:LabNotes/Microbiome-new/2010-5-2: Difference between revisions
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>Sam Chiang |
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*Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue). | *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. | *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. ( | *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=== | ===Library enrichment by PCR amplification=== |
Revision as of 15:05, 4 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
- 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. (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)x12 cycle -> 4C forever
- NOTE: the result should be about 500ng amplified DNA (Zymo manual)