Noi/NOTES/2012-7-31
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Preparation of sheared gDNA for N37 sample set[edit]
- 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
Sample IDs | Sample code on tubes | Original conc. (ng/ul) | Volume for 200ng (ul) |
EB (ul) | 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 |
- Sheared on 2012.08.01
Follow up the set up for Covaris shearing (From BioGem and Alan's note: [[1]]) 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
- 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