Sam:LabNotes/Microbiome-new/2010-12-29
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Nextera-library construction on E.coli SAG(WGA1027'10, WGA1103'10)
Background
- Reference link:
Sample preparation
- Eight E.coli SAGs will be used for library construction.
- Only four samples will be used in today's trial.
- Sample quantification info: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-17
- Thaw the tRNA carrier (stored in -80C). Use the "2"-tRNA(96ng/uL)
Trial-1(12-29-'10) Sample no. Sample name Conc.(ng/uL) Sample(uL) tRNA("2") H2O Total DNA(ng) --------------------------------------------------------------------------------------------------- NX01 WGA102710-(1-4)- 3hr 5.77 50.00 0.0 0.00 288.50ng NX02 WGA102710-(1-4)-10hr 1162.79 0.43 - 49.00 500.00ng NX03 WGA110310-(5)- 3hr 2.29 50.00 0.0 0.00 114.50ng NX04 WGA110310-(5)- 10hr 555.81 0.90 - 49.00 500.00ng
- I am trying not to added any tRNA carrier in for EtOH precipitation since the samples amount for short-amplification is around 100~300ng.
EtOH purification of MDA amplicons
- Add tRNA carrier(96ng/uL) into short-amplified MDA amplicon (to make total amount of 400ng).
- Total volume: 50uL.
- Add 2.5X volume 100% EtOH (125uL) + 0.1X volume 3M NaOAc (5uL) + 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(10ng)+ 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)x10 cycle -> 4C forever
- NOTE: The manual suggested only amplify 9 cycle. Since my starting amount is only 10ng (instead of 50ng), I amplify one more cycle (total 10cycle).
Ampure bead purification
- Following standard protocol with 0.7V volume AMpure beads.
- Nanodrop the purified DNA and stored it in -20C immediately.
Results
- 3uL of amplified product was validated using TBE-PAGE
- 3uL PCR product + 3uL 0.5X TBE + 3uL 6X loading dye.
File:Sam072410-Nextera lib.jpg
- I accidently over-run the gel (250V, 30min). I should only use 200V instead.
- The bands showed up but look smear.
- For the 10ng starting amount, 10cycle PCR amplification is not enough.
Extra-library amplification using rt-PCR
- Since there are 10uL template left from the 1st step of Nextera library construction. Only 5uL were used for PCR amplification.
- I am trying to use the other 5uL template(transposon tagged DNA) for amplification.
- Set up the reaction on cold block in EP-white PCR-strip tubes:
1rxn x2.2 rxn RT-PCR grade H2O 16.8 36.96 DNA lib (from last step) 5.0 - 2X Nextera PCR buf. 25.0 55.00 50X Nextera primier cocktail 1.0 2.20 - Illumina-compatible 50X Nextera adaptor 2 1.0 2.20 - could be stwitch to barcoded library (optional) Netera PCR enzyme 1.0 2.20 50X SYBR-green I 0.2 0.44 ------------------------------------------- 50.0 99.00/2.2=45 --- template 5uL NOTE: I use the "no.2" SYBR-green 50X Stop the reaction before the amplification curve reach pleatue
Results
- 3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(200V, 30min).
File:Sam072910-nextera lib amp-2nd amp.jpg
- The 90min-library showed good range between 200-400bp
- The 10hr-library is probably over-loaded on gel. I need to do the nanodrop on this amplicon and validate again.
- After AMpure amplification the concentration could be raised up becasued the totoal volume was changed from 50uL to 30uL (I used 30uL H2O to elute the purified DNA).
- The comparison of before and after AMpure bead purification showed no significatn loss with the purification method.