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