Arichard:Notebook/2013/September

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Revision as of 21:05, 17 September 2013 by >Andrew (→‎09/16/2013)
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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 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

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

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

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

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

  • 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

File:2013 09 12 midas alz pcr2.jpg

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

File:2013 09 16 midas alz pcr1.jpg