Sam:LabNotes/Microbiome-new/2010-12-29: Difference between revisions
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>Sam Chiang |
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*3uL of amplified product was validated using TBE-PAGE | *3uL of amplified product was validated using TBE-PAGE | ||
**3uL PCR product + 3uL 0.5X TBE + 3uL 6X loading dye. | **3uL PCR product + 3uL 0.5X TBE + 3uL 6X loading dye. | ||
[[File: | [[File:samxxxxxx-Nextera lib.jpg|250px]] | ||
='''Extra-library amplification using rt-PCR'''= | ='''Extra-library amplification using rt-PCR'''= |
Revision as of 00:11, 30 December 2010
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
- Total volume: 50uL.
- UV-decontamination the 100% EtOH, 3M NAOAc and GlycoBlue in crosslinker oven for 20min.
- 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.
- NOTE: White big pellets still showed in short-amplified samples which suggested their appearance has nothing to do with over-dose tRNA adding or the introduced contamination during Covaris shearing (in HMS by Sara). The white pellet may due to MDA regent content, either N6 primers or enzyme proteins.
Tagmentation reaction
- Set up tagmentation reaction on cold block:
1rxn 4.5rxn DNA template+H2O 15.0 - 5X Nextera Rxn buf.(LMW) 4.0 18.0 Nextera enzyme 1.0 4.5 ------------------------------------- 20.0 22.5/4.5 = 5uL -- template 15uL
- Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
- During the incubation, setup the tube and buffer of MiniElute columns.
- Reaction products were purified using MiniElute column
- MiniElute columns have lids which are better to avoid introduced contamination.
- The efficiency of MiniElute columns has been verified in Nextera-kit manual.
- Elute DNA in 11uL pre-warmed EB buffer.
Library enrichment by PCR amplification
- Set up the reaction on cold block in Eppendorf white-bottom PCR strip tubes:
1rxn 4.5rxn RT-PCR grade H2O 16.0 72.0 DNA lib (from last step) 5.0 - 2X Nextera PCR buf. 25.0 112.5 50X Nextera primier cocktail 1.0 4.5 - Illumina-compatible 50X Nextera adaptor 2 1.0 4.5 - Using barcoded adaptor (ID1 - ID4) Nextera PCR enzyme 1.0 4.5 20X Eva-green 1.0 4.5 ----------------------------------------- 50.0 202.5 uL /4.5= 45 --- 5uL template *Eva-green or SYBR-green acceptable range is from 0.2X~0.4X in rt-PCR reaction
- Briefly vortexing and centrifuging.
- Pre-cut the tubes
- Program "Nextera lib amplification with Nextera-PCR enzyme": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x15 cycle -> 4C forever
- Pick up sample around 9 cycles (for 50ng-template reactions).
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:Samxxxxxx-Nextera lib.jpg
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.