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= Preparation of sheared gDNA for N37 sample set = * Alice makes an appointment with James at Biogen '''Sample list and code on the tube''' * From the previous experiment, gDNA of the 10 tissues of N37 were diluted to 100ng/ul total volume 20ul. I used 12 ul for BSPP assays, and have 8ul left. * I need to check if there is some evaporation and need to bring total volume to 8ul with EB buffer. * I plan to shear 200ng of gDNA, so I will take 2ul of gDNA and add to 48ul of H2O. * The total volume reduced to ~5ul, I added 3ul to each tube to bring volume up to 8ul {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Sample code on tubes''' | align="center" style="background:#f0f0f0;"|'''Original conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume <br> for 200ng (ul)''' | width="60pt" align="center" style="background:#f0f0f0;"|'''EB (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | N37-Cerebellum (CRBL)||N37-1||~100||2.00||48.00||50.00 |- | N37-Colon||N37-2||~100||2.00||48.00||50.00 |- | N37-Frontal lobe (FL)||N37-3||~100||2.00||48.00||50.00 |- | N37-Heart||N37-4||~100||2.00||48.00||50.00 |- | N37-Small intestine (SI)||N37-5||~100||2.00||48.00||50.00 |- | N37-Liver||N37-6||~100||2.00||48.00||50.00 |- | N37-Lung||N37-7||~100||2.00||48.00||50.00 |- | N37-Skeletal muscle (SM)||N37-8||~100||2.00||48.00||50.00 |- | N37Pancrease||N37-9||~100||2.00||48.00||50.00 |- | N37Stomach||N37-10||~100||2.00||48.00||50.00 |}<br> * Sheared on 2012.08.01 '''Follow up the set up for Covaris shearing (From BioGem and Alan's note''': [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Methylome/2012-1-17]]) Target BP 400 Tube Micro Duty Factor 10% Peak Incident Power (w) 140 Cycle per Burst 200 Time (secs) 55 Volume 50ul Temperature (c) 7 Intensifier Yes * Since the total amount of DNA was quite low, so I verified them before preparing the sequencing library by PAGE (6% TBE gel) [[File:ZhangLab_2 2012-08-09 14hr 21min_shearedDNA_N37.jpg| 400px]] * 2012.08.07: I've talked to David from Eppendorf and ask him more detail about the reaction set up KAPA low-input protocol. He mentioned that most of the reaction set up for the low-input protocol are the same as the general protocol, but the shaking speed and time after mixing with reaction mix were optimized. * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-8-26
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