Arichard:Notebook/2013/September: Difference between revisions

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>Andrew
>Andrew
 
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* Alan has asked that we run gels on our libraries ready for sequencing, i.e., after size selection.
* 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|400px]]
[[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