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='''Nextera-library construction on single E.coli MDA amplicons(0706'10 MDA)'''= ==Background== *An E.coli-positive amplicon was found using 16S-genotyping method (from 0706'10 MDA, 90m-well_C). *I also made extention-MDA amplicon for this sample (additinoal 8hr amplification) *These two samples will be used for making sequencing libraries: **Sam070610-90m-C (0.3ng/uL, 50uL) **Sam070610-90m-C-ext (379ng/uL, 20uL) *The library construction will be performed using Nextera lib kit: **Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-5-2 ==Sample preparation== *Only use about 10ng DNA for starting amount (10ng is the total available amount for the sample Sam070610-90m-C) **Use all of remained 30uL for Sam070610-90m-C (0.3ng/uL, 50uL)=> (~10ng) **Make 1/100 dilution for Sam070610-90m-C-ext(379ng/uL, 20uL)=> 3.8ng/uL => Use 3uL of this diluted template (~10ng) *'''There is no need for doing DNA shearing because I am plan to use Nextera lib construction (Transposon-based)''' ===EtOH purification of MDA amplicons=== **Add 4.9uL 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(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|250px]] *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|300px]] *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.
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