Arichard:Notebook/2013/September: Difference between revisions

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>Andrew
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* 2 samples confirmed positive on gel. Nextera indexes #28 and #32 on first gel, none on second.
* 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|September 4, 2013 MIDAS, PGP1 chromosomes, PCR gel 1]]
[[File: 2013_09_04_midas_pgp1_pcrgel1.jpg|400px]]


[[File: 2013_09_04_midas_pgp1_pcrgel2.jpg|September 4, 2013 MIDAS, PGP1 chromosomes, PCR gel 2]]
[[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.
* Low success rate (2/14 compared to typically ~50%) may be due to incomplete removal of EtOH after precipitation before tagmentation.
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===09/06/2013===
===09/06/2013===


[[File: 2013_09_06_midas_alz_neurons_pcrgel1.jpg|September 6, 2013 MIDAS, Alzheimer's neurons, PCR gel 1]]
[[File: 2013_09_06_midas_alz_neurons_pcrgel1.jpg|400px]]


[[File: 2013_09_06_midas_alz_neurons_pcrgel2.jpg|September 6, 2013 MIDAS, Alzheimer's neurons, PCR gel 2]]
[[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).
* Some confusion over loading. Chris re-ran these gels on the following Monday (September 9).
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===09/09/2013===
===09/09/2013===


[[File: 2013_09_09_rerun_2013_09_06_midas_alz_neurons_pcrgel1.jpg|September 9, 2013 rerun of September 6 MIDAS, Alzheimer's neurons, PCR gel 1]]
[[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|September 9, 2013 rerun of September 6 MIDAS, Alzheimer's neurons, PCR gel 2]]
[[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.
* 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|September 9, 2013 MIDAS, PGP1 and Alzheimer's neurons, precut]]
[[File: 2013_09_09_midas_pgp1_and_alz_neurons_precut.jpg|400px]]


[[File: 2013_09_09_midas_pgp1_and_alz_neurons_cut.jpg|September 9, 2013 MIDAS, PGP1 and Alzheimer's neurons, cut]]
[[File: 2013_09_09_midas_pgp1_and_alz_neurons_cut.jpg|400px]]


* Size selected 200-600 bp
* Size selected 200-600 bp

Revision as of 22:57, 9 September 2013

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