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

[Protocol used]

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

File:2013 12 11 bulk cut1.jpg

File:2013 12 11 bulk cut2.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