Sam:LabNotes/Microbiome-new/2010-11-15
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2nd size selection of pooled Illumina library(short and long amplified E.coli SAGs -3rd test 11-07-'10)[edit]
Background[edit]
- Eight indexed indivudual libraries were pooled based on equal amount (20ng)
- The pool library will be size-selected again in order to get a narrower band for sequencing.
Procedures[edit]
2nd size selection(300~400bp)[edit]
- 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 File:Sam111510-TBE PAGE-post cut.jpg
- 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[edit]
- 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
- 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)[edit]
- The library pass the Sanger sequencing validation. 14/16 sequences results showed E.coli positive.
- 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