Arichard:Notebook/2013/September: Difference between revisions
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[[File:2013_09_17_midas_alz_pcr2.jpg|400px]] | [[File:2013_09_17_midas_alz_pcr2.jpg|400px]] | ||
===09/18/2013=== | |||
* 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=== | |||
* 2nd day of MIDAS: amplicon extraction, processing, tagmentation, and nextera PCR. | |||
===09/20/2013=== | |||
* Gel results from 09/19 MIDAS: | |||
[[File:2013_09_20_midas_alz_pcr1.jpg|400px]] | |||
[[File:2013_09_20_midas_alz_pcr2.jpg|400px]] | |||
* Called 8 positives samples in total. | |||
===09/23/2013=== | |||
* Size selected libraries from 9/17 and 9/20: | |||
[[File:2013_09_23_midas_alz_precut1.jpg|400px]] | |||
[[File:2013_09_23_midas_alz_cut1.jpg|400px]] | |||
[[File:2013_09_23_midas_alz_precut2.jpg|400px]] | |||
[[File:2013_09_23_midas_alz_cut2.jpg|400px]] | |||
[[File:2013_09_23_midas_alz_precut3.jpg|400px]] | |||
[[File:2013_09_23_midas_alz_cut3.jpg|400px]] | |||
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=== | |||
* 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=== | |||
* Could not call sufficient number of positives to process. Will try again tomorrow. | |||
===09/26/2013=== | |||
* MIDAS on ID# 25-00+ | |||
* Got trained on Technics PEIIB Planar Etcher in Nano3. | |||
===09/30/2013=== | |||
* 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|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:
- 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[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.