Noi/NOTES/2012-7-31: Difference between revisions
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>Noi (Created page with "http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar'''<br> = Preparation of sheared gDNA for N37 sample set = * Alice make an appointme...") |
>Noi mNo edit summary |
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[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]<br> | [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]<br> | ||
= Preparation of sheared gDNA for N37 sample set = | = Preparation of sheared gDNA for N37 sample set = | ||
* Alice | * Alice makes an appointment with James at Biogen | ||
'''Sample list and code on the tube''' | '''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. | * 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 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. | * 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 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Sample IDs''' | | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | ||
Line 33: | Line 34: | ||
|- | |- | ||
| N37Stomach||N37-10||~100||2.00||48.00||50.00 | | N37Stomach||N37-10||~100||2.00||48.00||50.00 | ||
|} | |}<br> | ||
'''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]] | * 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 | Target BP 400 | ||
Tube Micro | Tube Micro | ||
Line 44: | Line 46: | ||
Temperature (c) 7 | Temperature (c) 7 | ||
Intensifier Yes | 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 |
Latest revision as of 09:03, 28 August 2012
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