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='''Mouse gut bacteria SAG ("c5") preparation - Library amplification from ligation products'''= ==Objective== *I prepared two Illumina libraries using limited MDA (2hr incubation) strategy, but only one of the samples was sequenced. **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2 *I still have the ligation product left from this sample. I will amplify it using N2-index primer and give it to Alan for sequencing. ==Illumina library amplification references== *Ligation product amplification with N2-indexed primer **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-07_exp1 *AMpure bead purification **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-14 *2nd size-selection **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-15 ==Procedures== *Prepare ice box. Clean the PCR hood. Set up realtime PCR program. *UV the following tubes: #PCR tube strip x1(Bio-rad) cap x1(Bio-rad) #0.5mL tube x1 (for master mix) #Cold block *Centrifuge the ligation products. 2min, 10000rpm, RT. Leave on ice. *Thaw the primers:N2-indexed-Fs(100uM) and N2-indexed-10R(100uM). Diluted them into 10uM (5uL stock sol. + 45uL H2O) *Thaw 2X Phusion HF enzyme mix *Setup the qPCR program. *Lib-amp PCR(100uL): (modified from Dr.Zhang recipe) 1rxn 2.5 rxn H2O 34 85.0 uL Adapter ligated DNA 10 - uL PCR_F(10uM) 2 5.0 uL PCR_R.N2 Index(10uM) 2 - uL --- using N2-indexed-primer "10" 20X EVA Green 2 5.0 uL 2X Phusion HF MM 50 125.0 uL ------------------ 100 220.0/2.5=88 --- template 10uL, Index primer(R) 2uL *NOTE1: I also amplify another library c6 to balance with c5. *NOTE2: '''N2-index-(10)R''' primer was used for both C5 and C6 libraries. *The reactions tubes were pre-cut before incubation. **Using new two-step PCR program. qPCR was performed using Eppendorf realplex machine: 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles -> (98C 10sec -> 72C 20sec) x 10 cycles -> 72C 3min -> hold 15C. *I will expect the stop the reactions around 15th~16th cycle or the monitored dif. of Flouresscensce reach 14000. Depends on which one reach first. *Aliquote 3uL of amplified library for TBE-PAGE validation later. *Use AMpure beads(1V, 100uL) to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation. **In AMpure bead manual, 1.8V beads are recommended. *For TBE-PAGE validation. Different sample amount were loaded since purified sample is much more concentrated, and I don't want to waste it on gel loading. **Un-purified samples(3uL) and purified sample(1uL). *TBE-PAGE **Unpurified sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer **Puriried samplewell : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer **Ladder well : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer **Gel was run at 200V for 30min. **Use 3uL SYBR-gold with regular stainig(5~10min) with foil protection ==Results== [[File:042811-amp lib c5 and c6.png|350px]] *The AMpure beads can remove unwanted size under 100bp, but there still are over-size(>500bp) molecular in the library c5. ==2nd size selection (05-01-'11)== *The purified library is ~20uL. Split sample in two 10uL volume and run 10uL sample/well on a 5-well TBE-PAGE **Sample well: 10uL H2O + 10uL 6X loading dye + 10uL DNA sample **Ladder well: 20uL H2O + 10uL 6X loading dye + 0.5uL Low mass ladder/1kb plus ladder **Run at 200V for 30min. Long staining (15min with foil wrap protection). **Band size between 350~450bp was cut for c5 lib samples in both wells. [[File:050111-c5 library precut.png|350px]] [[File:050111-c5 library post-cut.png|370px]] *Shred the gel slice using pole-punctured 0.5mL non-sticky tubes. Centrifuge at 14,000rpm for 3min. *Incubate the gel slice mixture with 500uL clean 1X TE at 37C for 2hr on the vortexer. *Pellet the gel suspension at 14,000rpm for 1.5min at RT. *Transfer the supernatant onto Nanosap filter. Centrifuge at 14,000rpm for 1.5min at RT. **NOTE: I got ~350uL supernatant from each of samples. *Transfer the filtrate into clean 1.5-mL non-sticky tubes. *EtOH precipitation: 2.5V 100%EtOH (875uL), 0.1V 3M NaOAc(35uL), 1.5uL Glycoblue. Mix well by inverting and incubate at -80C for 25min *Centrifuge at 10,000xg 4C for 25min. Wash pellet with 750uL 75% EtOH, centrifuge at 10,000xg 4C for 5min. Air dry 5min. Elute the pellet in 15uL H2O. Pool eluted DNA together from two samples => 15X2 = 30uL. *Labeled the library as "'''HC-MDA-Mouse-2h-C5-Mar02'''"
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