Arichard:Notebook/2013/September

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Revision as of 22:55, 9 September 2013 by >Andrew
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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.

September 4, 2013 MIDAS, PGP1 chromosomes, PCR gel 1

September 4, 2013 MIDAS, PGP1 chromosomes, PCR gel 2

  • 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

September 6, 2013 MIDAS, Alzheimer's neurons, PCR gel 1

September 6, 2013 MIDAS, Alzheimer's neurons, PCR gel 2

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

09/09/2013

September 9, 2013 rerun of September 6 MIDAS, Alzheimer's neurons, PCR gel 1

September 9, 2013 rerun of September 6 MIDAS, Alzheimer's neurons, PCR gel 2

  • 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.

September 9, 2013 MIDAS, PGP1 and Alzheimer's neurons, precut

September 9, 2013 MIDAS, PGP1 and Alzheimer's neurons, cut

  • Size selected 200-600 bp