Noi/NOTES/2014-10-29: Difference between revisions
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'''NOTE:''' The SeqCap Epi probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap Epi probe pool, Roche NimbleGen recommends aliquoting the SeqCap Epi probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles.<br> | '''NOTE:''' The SeqCap Epi probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap Epi probe pool, Roche NimbleGen recommends aliquoting the SeqCap Epi probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles.<br> | ||
'''Label PCR tube:''' Hg19 UMR v1 141029 | '''Label PCR tube:''' Hg19 UMR v1 141029 | ||
'''Location:''' -20C, in | '''Location:''' -20C, in original box within Noi Library prep kit box. | ||
I got total 16 tubes of 4.5ul aliquot. The instruction said it's normal to get extra volume of the probe. | |||
'''2. For sequencing library preparation''' | '''2. For sequencing library preparation''' | ||
# Take a small aliquot the left over of the probe pool after a single-use aliquots for 12 rxn for library prep using ClonTech DNA SMRT kit | # Take a small aliquot the left over of the probe pool after a single-use aliquots for 12 rxn for library prep using ClonTech DNA SMRT kit | ||
* There is ~1.5-2ul of the probe left in the tube after aliquot for single-use. I added 4ul of H2O to increase the volume. I then took 1ul for QUbit ssDNA quantification. | |||
* Qubit result = ng/ul --> original concentration from Nimblegen should be ~ ng/ul. | |||
* I use XXng or ul for DNA SMART ChIP seq experiment |
Revision as of 18:24, 29 October 2014
Generation of sequencing library of a customized SeqCap EZ Epi Probe Pool
- 2014-10-29: I am expecting the package of a customized SeqCap EZ Epi probe pool that Dr. Zhang ordered for target enrichment of the blood UMRs from all WGBS libraries
- Probe information:
- Design name: 140923_HG19_UMR_v1_Epi_HX1
- Volume: 54ul
Things to do upon receive the probe pool
1. Recommended by instruction in CHAPTER2:
- Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap Epi probe pool to avoid multiple freeze/thaw cycles or potential accidental contamination:
- If frozen, thaw the tube of SeqCap Epi probe pool on ice.
- Vortex the SeqCap Epi probe pool for 3 seconds.
- Centrifuge the tube of SeqCap Epi probe pool at 10,000 x g for 30 seconds to ensure that the liquid is at the bottom of the tube before opening the tube.
- Aliquot the SeqCap Epi probe pool into single-use aliquots (4.5 µl/aliquot) in 0.2 ml PCR tubes (or 96-well plates if following the higher throughput protocol described in Appendix C) and store at -15 to -25°C until use. The presence of some residual volume after dispensing all single-use aliquots is normal.
- When ready to perform the experiment, thaw the required number of single-use SeqCap Epi probe pool aliquots on ice.
NOTE: The SeqCap Epi probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap Epi probe pool, Roche NimbleGen recommends aliquoting the SeqCap Epi probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles.
Label PCR tube: Hg19 UMR v1 141029 Location: -20C, in original box within Noi Library prep kit box. I got total 16 tubes of 4.5ul aliquot. The instruction said it's normal to get extra volume of the probe.
2. For sequencing library preparation
- Take a small aliquot the left over of the probe pool after a single-use aliquots for 12 rxn for library prep using ClonTech DNA SMRT kit
- There is ~1.5-2ul of the probe left in the tube after aliquot for single-use. I added 4ul of H2O to increase the volume. I then took 1ul for QUbit ssDNA quantification.
- Qubit result = ng/ul --> original concentration from Nimblegen should be ~ ng/ul.
- I use XXng or ul for DNA SMART ChIP seq experiment