Sam:LabNotes/Microbiome-new/2010-11-15: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang |
||
Line 24: | Line 24: | ||
*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
- 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
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