Arichard:Notebook/2013/September: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Andrew
(Created page with "==September, 2013== ===09/03/2013=== Chris and I will be taking responsibility for MIDAS from here on. Started overnight MDA with Jeff and Chris observing. Loaded 8 arrays ...")
 
>Andrew
 
(18 intermediate revisions by the same user not shown)
Line 9: Line 9:
* 2 conditions:
* 2 conditions:
** 4 arrays with protease (trypsin)
** 4 arrays with protease (trypsin)
** 4 arrays, standard protocol except no freeze thaw
** 4 arrays, [[Arichard:Protocols/MDA on microarray 2013 09 05|standard protocol]] except no freeze thaw


Did not image prior to ALS because chromosomes were expected in every well due to high concentration.
Did not image prior to ALS because chromosomes were expected in every well due to high concentration.
Line 30: Line 30:
*** Total = 11.2 ul
*** Total = 11.2 ul


* No protease condition received 11.2 ul standard MDA master mix
* No protease condition received 11.2 ul [[Arichard:Protocols/MDA on microarray 2013 09 05|standard MDA master mix]]


===09/04/2013===
===09/04/2013===
Line 38: Line 38:
1 NTC showed large amount of amplification during blue/orange PCR, more than all other samples. Reagent contamination would have been seen in all samples.
1 NTC showed large amount of amplification during blue/orange PCR, more than all other samples. Reagent contamination would have been seen in all samples.


* 2 samples confirmed positive on gel. Nextera indexes #28 and #32.
* 2 samples confirmed positive on gel. Nextera indexes #28 and #32 on first gel, none on second.
 
[[File: 2013_09_04_midas_pgp1_pcrgel1.jpg|400px]]
 
[[File: 2013_09_04_midas_pgp1_pcrgel2.jpg|400px]]
 
* Low success rate (2/14 compared to typically ~50%) may be due to incomplete removal of EtOH after precipitation before tagmentation.


===09/05/2013===
===09/05/2013===


Chris started overnight MDA with Jeff and I observing. Loaded 8 arrays with neuronal nuclei.
Chris started overnight MDA with Jeff and I observing. Loaded 8 arrays with neuronal nuclei.
===09/06/2013===
[[File: 2013_09_06_midas_alz_neurons_pcrgel1.jpg|400px]]
[[File: 2013_09_06_midas_alz_neurons_pcrgel2.jpg|400px]]
* Some confusion over loading. Chris re-ran these gels on the following Monday (September 9).
===09/09/2013===
[[File: 2013_09_09_rerun_2013_09_06_midas_alz_neurons_pcrgel1.jpg|400px]]
[[File: 2013_09_09_rerun_2013_09_06_midas_alz_neurons_pcrgel2.jpg|400px]]
* Same as initial gels, so the loading was correct after all. Positive indexes called: 43 and 45 from the first gel, 46 and 47 from the second gel.
[[File: 2013_09_09_midas_pgp1_and_alz_neurons_precut.jpg|400px]]
[[File: 2013_09_09_midas_pgp1_and_alz_neurons_cut.jpg|400px]]
* Size selected 200-600 bp
===09/10/2013===
* Started MIDAS run with fresh nuclei. (Chris used the same ones last week).
** Sample #102 (Alzheimer's)
** 8 arrays
** Latest MIDAS protocol
I forgot to stain the cells with 1X SYBR Green prior to loading, so I was not able to take loading images. We will have to call positive wells based on fluorescence alone.
===09/11/2013===
* Finished PCR and gel.
** Loaded gel with 10 ul (instead of 5 ul) + 3 ul 6X loading dye. (10% of PCR reaction).
** Chris and I called 6 positive samples
*** First gel: Samples 1, 3, and 5. Indexes 25, 27, and 29, respect.
*** Second gel: Samples 3, 4, and 5. Indexes 27, 28, and 29, respect.
[[File:2013_09_12_midas_alz_pcr1.jpg|400px]]
[[File:2013_09_12_midas_alz_pcr2.jpg|400px]]
===09/12/2013===
* Bead purified positive samples called yesterday.
* Chris is starting MIDAS run today. Same samples:
** Alzheimer's neurons, sample ID 102
===09/13/2013===
* Friday the 13th.
** Spooky
* Chris is running 2nd day of MIDAS protocol.
** 15 samples + 1 NTC (sample #16)
===09/16/2013===
* Chris and I ran analytical gels for Chris's MIDAS run (from 09/12 and 9/13).
[[File:2013_09_16_midas_alz_pcr1.jpg|400px]]
[[File:2013_09_16_midas_alz_pcr2.jpg|400px]]
* Total positive samples from last week = 11
[[File:2013_09_16_midas_alz_precut1.jpg|400px]]
[[File:2013_09_16_midas_alz_precut2.jpg|400px]]
[[File:2013_09_16_midas_alz_cut1.jpg|400px]]
[[File:2013_09_16_midas_alz_cut2.jpg|400px]]
===09/17/2013===
* Extracted and processed MIDAS amplicons
[[File:2013_09_17_midas_alz_pcr1.jpg|400px]]
[[File:2013_09_17_midas_alz_pcr2.jpg|400px]]
===09/18/2013===
* Received 4 new samples from Gwen in the Chun lab, neuronal and non-neuronal from with and without AD. See sequencing spreadsheet link under Jeff's namespace.
* Started MIDAS with new sample, neuronal AD nuclei:
** Sample ID #25-00 + (neuronal)
** 34,000 cells in 250 ul --> 136 cells/ul/
* 1 in 10 dilution gave good loading.
===09/19/2013===
* 2nd day of MIDAS: amplicon extraction, processing, tagmentation, and nextera PCR.
===09/20/2013===
* Gel results from 09/19 MIDAS:
[[File:2013_09_20_midas_alz_pcr1.jpg|400px]]
[[File:2013_09_20_midas_alz_pcr2.jpg|400px]]
* Called 8 positives samples in total.
===09/23/2013===
* Size selected libraries from 9/17 and 9/20:
[[File:2013_09_23_midas_alz_precut1.jpg|400px]]
[[File:2013_09_23_midas_alz_cut1.jpg|400px]]
[[File:2013_09_23_midas_alz_precut2.jpg|400px]]
[[File:2013_09_23_midas_alz_cut2.jpg|400px]]
[[File:2013_09_23_midas_alz_precut3.jpg|400px]]
[[File:2013_09_23_midas_alz_cut3.jpg|400px]]
Sequencing sample IDs are listed on gel images. Libraries are named by the date of the analytical PCR gel to avoid confusion. Samples from 9/17 are cerebellum sample ID #102, samples from 9/20 are cortex sample ID #25-00.
===09/24/2013===
* Chris started MIDAS run: ID# 25-00+, Alzheimer's neuronal nuclei from 9/17/2013.
** Low loading concentration.
** Did not save loading images. Will have to call positive wells by fluorescence increase.
** Chris reported no more than 10 cells observed.
===09/25/2013===
* Could not call sufficient number of positives to process. Will try again tomorrow.
===09/26/2013===
* MIDAS on ID# 25-00+
* Got trained on Technics PEIIB Planar Etcher in Nano3.
===09/30/2013===
* Alan has asked that we run gels on our libraries ready for sequencing, i.e., after size selection.
[[File:2013_09_30_midas_alz_lib.jpg|400px]]

Latest revision as of 19:32, 1 October 2013

September, 2013[edit]

09/03/2013[edit]

Chris and I will be taking responsibility for MIDAS from here on.

Started overnight MDA with Jeff and Chris observing. Loaded 8 arrays with PGP-1 chromosomes.

  • 2 conditions:

Did not image prior to ALS because chromosomes were expected in every well due to high concentration.

  • Modification to standard protocol for protease treatment:
    • Load 3 ul chromosomes. Add coverslip, let sit 10 min @ RT
    • Load 3 ul 0.25% trypsin (1X) into protease condition arrays. Cover and let sit 5 min @ RT
    • Load 4.5 ul ALS into all arrays. Cover and incubate 10 min @ 40 degC in thermocycler with plate adapter
    • Load 4.5 ul NS into all arrays.
      • Protocol based on Quake haplotype paper (Fan et al. 2010 NBT)
  • Protease condition master mix(per rxn/array):
    • 15 ul template
    • 1 ul 1 mM N6*
    • 1 ul 25 mM dNTP (Epicentre)
    • 2.3 ul 10X Phi29 buffer
    • 0.1 ul 50X SYBR Green
    • 1 ul 23X protease inhibitor (cOmplete)
    • 1 ul H2O
      • Total = 11.2 ul

09/04/2013[edit]

Extracted and processed 14 samples + 2 NTCs (no extraction)

1 NTC showed large amount of amplification during blue/orange PCR, more than all other samples. Reagent contamination would have been seen in all samples.

  • 2 samples confirmed positive on gel. Nextera indexes #28 and #32 on first gel, none on second.

File:2013 09 04 midas pgp1 pcrgel1.jpg

File:2013 09 04 midas pgp1 pcrgel2.jpg

  • Low success rate (2/14 compared to typically ~50%) may be due to incomplete removal of EtOH after precipitation before tagmentation.

09/05/2013[edit]

Chris started overnight MDA with Jeff and I observing. Loaded 8 arrays with neuronal nuclei.

09/06/2013[edit]

File:2013 09 06 midas alz neurons pcrgel1.jpg

File:2013 09 06 midas alz neurons pcrgel2.jpg

  • Some confusion over loading. Chris re-ran these gels on the following Monday (September 9).

09/09/2013[edit]

File:2013 09 09 rerun 2013 09 06 midas alz neurons pcrgel1.jpg

File:2013 09 09 rerun 2013 09 06 midas alz neurons pcrgel2.jpg

  • Same as initial gels, so the loading was correct after all. Positive indexes called: 43 and 45 from the first gel, 46 and 47 from the second gel.

File:2013 09 09 midas pgp1 and alz neurons precut.jpg

File:2013 09 09 midas pgp1 and alz neurons cut.jpg

  • Size selected 200-600 bp

09/10/2013[edit]

  • Started MIDAS run with fresh nuclei. (Chris used the same ones last week).
    • Sample #102 (Alzheimer's)
    • 8 arrays
    • Latest MIDAS protocol

I forgot to stain the cells with 1X SYBR Green prior to loading, so I was not able to take loading images. We will have to call positive wells based on fluorescence alone.

09/11/2013[edit]

  • Finished PCR and gel.
    • Loaded gel with 10 ul (instead of 5 ul) + 3 ul 6X loading dye. (10% of PCR reaction).
    • Chris and I called 6 positive samples
      • First gel: Samples 1, 3, and 5. Indexes 25, 27, and 29, respect.
      • Second gel: Samples 3, 4, and 5. Indexes 27, 28, and 29, respect.

File:2013 09 12 midas alz pcr1.jpg

File:2013 09 12 midas alz pcr2.jpg

09/12/2013[edit]

  • Bead purified positive samples called yesterday.
  • Chris is starting MIDAS run today. Same samples:
    • Alzheimer's neurons, sample ID 102

09/13/2013[edit]

  • Friday the 13th.
    • Spooky
  • Chris is running 2nd day of MIDAS protocol.
    • 15 samples + 1 NTC (sample #16)

09/16/2013[edit]

  • Chris and I ran analytical gels for Chris's MIDAS run (from 09/12 and 9/13).

File:2013 09 16 midas alz pcr1.jpg

File:2013 09 16 midas alz pcr2.jpg

  • Total positive samples from last week = 11

File:2013 09 16 midas alz precut1.jpg

File:2013 09 16 midas alz precut2.jpg

File:2013 09 16 midas alz cut1.jpg

File:2013 09 16 midas alz cut2.jpg

09/17/2013[edit]

  • Extracted and processed MIDAS amplicons

File:2013 09 17 midas alz pcr1.jpg

File:2013 09 17 midas alz pcr2.jpg

09/18/2013[edit]

  • Received 4 new samples from Gwen in the Chun lab, neuronal and non-neuronal from with and without AD. See sequencing spreadsheet link under Jeff's namespace.
  • Started MIDAS with new sample, neuronal AD nuclei:
    • Sample ID #25-00 + (neuronal)
    • 34,000 cells in 250 ul --> 136 cells/ul/
  • 1 in 10 dilution gave good loading.

09/19/2013[edit]

  • 2nd day of MIDAS: amplicon extraction, processing, tagmentation, and nextera PCR.

09/20/2013[edit]

  • Gel results from 09/19 MIDAS:

File:2013 09 20 midas alz pcr1.jpg

File:2013 09 20 midas alz pcr2.jpg

  • Called 8 positives samples in total.

09/23/2013[edit]

  • Size selected libraries from 9/17 and 9/20:

File:2013 09 23 midas alz precut1.jpg

File:2013 09 23 midas alz cut1.jpg

File:2013 09 23 midas alz precut2.jpg

File:2013 09 23 midas alz cut2.jpg

File:2013 09 23 midas alz precut3.jpg

File:2013 09 23 midas alz cut3.jpg

Sequencing sample IDs are listed on gel images. Libraries are named by the date of the analytical PCR gel to avoid confusion. Samples from 9/17 are cerebellum sample ID #102, samples from 9/20 are cortex sample ID #25-00.

09/24/2013[edit]

  • Chris started MIDAS run: ID# 25-00+, Alzheimer's neuronal nuclei from 9/17/2013.
    • Low loading concentration.
    • Did not save loading images. Will have to call positive wells by fluorescence increase.
    • Chris reported no more than 10 cells observed.

09/25/2013[edit]

  • Could not call sufficient number of positives to process. Will try again tomorrow.

09/26/2013[edit]

  • MIDAS on ID# 25-00+
  • Got trained on Technics PEIIB Planar Etcher in Nano3.

09/30/2013[edit]

  • Alan has asked that we run gels on our libraries ready for sequencing, i.e., after size selection.

File:2013 09 30 midas alz lib.jpg