Arichard:Notebook/2013/December: Difference between revisions
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All samples were stained with PI instead of DAPI due to concerns about the DNA damage upon excitation. This was unnecessary, however, as DAPI uses a 405 nm Ex wavelenth, which is not UV and nowhere near the absorption peak for thymine dimerization (254 nm). | All samples were stained with PI instead of DAPI due to concerns about the DNA damage upon excitation. This was unnecessary, however, as DAPI uses a 405 nm Ex wavelenth, which is not UV and nowhere near the absorption peak for thymine dimerization (254 nm). | ||
===12/11/2013=== | |||
* Lysed cells in Rui's hypotonic lysis buffer | |||
Hypotonic lysis buffer: | |||
Make 1M MgCl2 solution, filter with 0.22um filter | |||
Make 10ml 10X NP40 lysis buffer [100mM Tris.HCl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004] | |||
Make 1ml 1X lysis buffer + 40ul protease (0.2Au/ml final) | |||
Controls introduced at first step. | |||
Positive control was 1 ul of 5.6 ng/ul E. coli gDNA. | |||
Negative control was NTC. | |||
* Digested 3 hours at 50 degC. | |||
* EtOH precipitated and resuspended in 7 ul H2O. Some cells were very large, leading to excess volume. Of these, only 7 ul was used for Nextera. | |||
* Followed standard protocol from then on: | |||
** 7 ul sample, 2 ul 5X HMW buffer, 1 ul 1:50 Nextera. 5 min @ 55 degC. | |||
** Protease | |||
** Exo- | |||
** PCR with adapters | |||
** PCR with primers | |||
** Ampure XP | |||
** Size select and gel extract | |||
** EtOH precip | |||
[[File: 2013_12_11_bulk_nxtpcr.jpg|400px]] | |||
[[File: 2013_12_11_bulk_precut1.jpg|400px]] | |||
[[File: 2013_12_11_bulk_precut2.jpg|400px]] | |||
[[File: 2013_12_11_bulk_cut1.jpg|400px]] | |||
[[File: 2013_12_11_bulk_cut2.jpg|400px]] | |||
Libraries submitted to Alan: | |||
* 7 pools | |||
** 1: AR_bulk_25,27_Dec11 | |||
** 2: AR_bulk_26_Dec11 | |||
** 3: AR_bulk_28,30_Dec11 | |||
** 4: AR_bulk_29_Dec11 | |||
** 5: AR_bulk_31,36_Dec11 | |||
** 6: AR_bulk_32_Dec11 | |||
** 7: AR_bulk_37,38_Dec11 |
Latest revision as of 22:12, 16 December 2013
December, 2012[edit]
12/02/2013[edit]
Meeting with Ben Siddoway and Gwen Kaeser.
Discussed possible quality control measures.
- Neuronal nuclei come from three brain banks:
- UC Irvine
- Maryland
- Wash U
12/03/2013[edit]
Shadowed Gwen for neuronal nuclei isolation
We decided to use the same samples, but take samples along the way for bulk prep (Nextera). Female preferred so that presence of Y chromosome can be used as a marker for contamination by myself.
Bulk sorted 3 types, all NeuN+
- 1864+, female Down's from ???.
- 1568+, female AD from Maryland.
- 61788+, female AD from Wash U. (New patient)
Notes
- Started with 0.095 g 1568, 0.14 g 61788, and 0.3 g 1864.
- Filtered with 40 um BD Falcon filter.
- Fixed 25 min, not 30.
Time point samples (3) for bulk prep on each sample (3) (9 total):
- 1 = After filter
- 2 = Before FACS
- 3 = After FACS
All samples were stained with PI instead of DAPI due to concerns about the DNA damage upon excitation. This was unnecessary, however, as DAPI uses a 405 nm Ex wavelenth, which is not UV and nowhere near the absorption peak for thymine dimerization (254 nm).
12/11/2013[edit]
- Lysed cells in Rui's hypotonic lysis buffer
Hypotonic lysis buffer: Make 1M MgCl2 solution, filter with 0.22um filter Make 10ml 10X NP40 lysis buffer [100mM Tris.HCl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004] Make 1ml 1X lysis buffer + 40ul protease (0.2Au/ml final)
Controls introduced at first step. Positive control was 1 ul of 5.6 ng/ul E. coli gDNA. Negative control was NTC.
- Digested 3 hours at 50 degC.
- EtOH precipitated and resuspended in 7 ul H2O. Some cells were very large, leading to excess volume. Of these, only 7 ul was used for Nextera.
- Followed standard protocol from then on:
- 7 ul sample, 2 ul 5X HMW buffer, 1 ul 1:50 Nextera. 5 min @ 55 degC.
- Protease
- Exo-
- PCR with adapters
- PCR with primers
- Ampure XP
- Size select and gel extract
- EtOH precip
File:2013 12 11 bulk nxtpcr.jpg
File:2013 12 11 bulk precut1.jpg
File:2013 12 11 bulk precut2.jpg
Libraries submitted to Alan:
- 7 pools
- 1: AR_bulk_25,27_Dec11
- 2: AR_bulk_26_Dec11
- 3: AR_bulk_28,30_Dec11
- 4: AR_bulk_29_Dec11
- 5: AR_bulk_31,36_Dec11
- 6: AR_bulk_32_Dec11
- 7: AR_bulk_37,38_Dec11