Sam:LabNotes/Microbiome-new/2010-11-15: Difference between revisions

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>Sam Chiang
>Sam Chiang
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*EtOH precipitation: 2.5V EtOH, 0.1V 3M NaOAc, 1.5uL Glycoblue. Mix well by inverting and incubate at -80C for 25min or more.
*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.
*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===
===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.
*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|400px]]

Revision as of 08:34, 16 November 2010

2nd size selection of pooled Illumina library(short and long amplified E.coli SAGs -3rd test 11-07-'10)

Background

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.
File:Sam111510-TBE PAGE.jpg  File:Sam111510-TBE PAGE-post cut.jpg
  • Sine 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