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='''2nd size selection of pooled Illumina library(short and long amplified E.coli SAGs -3rd test 11-07-'10)'''= ==Background== *Eight indexed indivudual libraries were pooled based on equal amount (20ng) **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-14 *The pool library will be size-selected again in order to get a narrower band for sequencing. ==Procedures== ===2nd size selection(300~400bp)=== *The pooled library is ~40uL. The pooled library was seperated into two tube (20uL/tube) for size-selection using 5-well TBE-PAGE. **Sample well: 10uL H2O + 10uL 6X loading dye + 20uL DNA sample **Ladder well: 30uL 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 '''300~400bp''' was cut in both pooled lib samples. **'''The size-selected regions should be the same regions I used for semi-quantification by TBE-PAGE from 11-14-'10''' [[File:sam111510-TBE PAGE.jpg|400px]] [[File:sam111510-TBE PAGE-post cut.jpg|450px]] *Since the estimated DNA amount in pooled lib is quite low (around 20x8=160ng => 160/2 = 80ng per well), I add 3.2uL tRNA(96ng/uL) to make the total nucleotide to 400ng before DNA recovery from gel-slice. *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 EtOH, 0.1V 3M NaOAc, 1.5uL Glycoblue. Mix well by inverting and incubate at -80C for 25min or more. *Centrifuge at 10,000xg 4C for 25min. Wash pellet with 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. ===TBE-PAGE semi-quantification=== *Since the EtOH-precipitation purified DNA is not able to be quantified by Nanodrop directly. I decided to use TBE-PAGE for another quantication. Also I can validate the result of size-selected library. [[File:sam111510-TBE PAGE -semi-quant.jpg|300px]] *The size selected libaray is confirmed around 300~400bp. *The estimated concentration of 2nd size-selected lib. is '''~1.76 ng/uL'''(by PAGE gel semi-quantification). The total yield of library is 1.76*30= 52.8 ng **Starting DNA amount: 20ng*8=160ng. Since I only size selected the partial region(~1/3 of whole smear), actually the DNA recovery rate is pretty high in the current experiment. *The library is ready for cloning Sanger sequencing validation. ===Follow up: AMpure bead purification (11-19-'10)=== *The library pass the Sanger sequencing validation. 14/16 sequences results showed E.coli positive. **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-16_exp1 *Since the library still carry short(~50bp) fragments (shown on PAGE), I did an additional AMpure bead purification to remove these short fragments: **Starting amount is set up as 30uL, I use 1V of AMpure beads(30uL) to bind on library DNA. **Wash with 200uL 70% EtOH twice, air dry 2min. **Elute DNA in 20uL pre-warmed H2O. *Library was submitted to Alan for sequencing. Library tag: '''HC-MDA-Ecoli-SAGs(Ind1to8)-Nov15'''
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