Sam:LabNotes/Microbiome-new/2010-5-2
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Nextera (Epicentre) kit - Illumina sequencing library construction[edit]
Background[edit]
- 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[edit]
- Two E.coli MDA amplicons (made on 03-28-'10)were used for the current tests
- Only use about 50ng DNA for starting amount
- QUBIT-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[edit]
EtOH purification of MDA amplicons[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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).
Results[edit]
- 3uL of amplified product was validated using TBE-PAGE
- 3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
- Run at 200V, 30min on a 10-well TBE-PAGE (Use Invitrogen premade gel).
- Long staing (15mins) with foil cover.
- Picture was taken at exposure time 4.096sec
File:Sam050210-nextera lib.jpg Concentration: 2h-Ecoli MDA(50ng) 10h-Ecoli MDA(50ng) QUBIT-HS (ng/uL) 17.0 15.4
- For using Low-molecular-weight(LMW) fragmentation buffer, the expeced size will be around 200-500bp. The result in gel picture support this point.
- There still have some smear tail above 500bp. I am curious if it is caused by over PCR amplification.
- Although the amplfication cycle (12 cycles) is suggested in protocol, I am inclined to test the result with 10cycles amplification using the remained 5uL templates.
- A size selection may be necessary for removing large fragments.
- NOTE: Condition summary
- Starting material: 50ng MDA amplicon (non-sheared, but EtOH purified).
- Using Zymo-DNA cleaning column
- 12cycle amplification (5uL template in 50uL rxn volume).
- The similar observation (over-sized libraries) was found on Epicenter's website
File:Nextera kit - oversize of libraries.jpg