Arichard:Notebook/2013/September: Difference between revisions
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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| | [[File: 2013_09_04_midas_pgp1_pcrgel1.jpg|400px]] | ||
[[File: 2013_09_04_midas_pgp1_pcrgel2.jpg| | [[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| | [[File: 2013_09_06_midas_alz_neurons_pcrgel1.jpg|400px]] | ||
[[File: 2013_09_06_midas_alz_neurons_pcrgel2.jpg| | [[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| | [[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| | [[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| | [[File: 2013_09_09_midas_pgp1_and_alz_neurons_precut.jpg|400px]] | ||
[[File: 2013_09_09_midas_pgp1_and_alz_neurons_cut.jpg| | [[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:
- 4 arrays with protease (trypsin)
- 4 arrays, standard protocol except no freeze thaw
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
- No protease condition received 11.2 ul standard MDA master mix
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