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===Size selection=== *20uL PCR product samples were run on 5-well PAGE (200V, 30min). **Sample well: 20uL sample + 10uL 6X loading dye + 10uL RT-PCR grade H2O **Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL RT-PCR grade H2O [[File:sam110810-lib.jpg|400px]] [[File:sam110810-lib postcut.jpg|400px]] Ligation product Ligation product (post gel-cut) *I still trying to continue the library construction this time. Also I add four samples which are not completed from 10-25-'10's test. Ref. link: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-10-24 New ID HC110910-1 HC110910-2 HC110910-3 HC110910-4 HC110910-5 HC110910-6 HC110910-7 HC110910-8 ---------------------------------------------------------------------------------------------------------------- Original ID SAM1 SAM2 SAM3 SAM4 1S 2S 1L 2L Original DATE 110610 110610 110610 110610 102510 102510 102510 102510 Lib Construction *For all of short-amplified samples I add 4.5uL tRNA(96ng/uL) to make the total nucleotide to 500ng 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. **NOTE: I did a over-night incubation for this step. *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: Usually the large gel slice get fewer supernatant. I got 350uL for the long-amplified samples and 300uL for the short-amplified 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 20uL H2O.
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