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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: ** 4 arrays with protease (trypsin) ** 4 arrays, [[Arichard:Protocols/MDA on microarray 2013 09 05|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 [[Arichard:Protocols/MDA on microarray 2013 09 05|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|400px]] [[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. ===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|400px]] [[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). ===09/09/2013=== [[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|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. [[File: 2013_09_09_midas_pgp1_and_alz_neurons_precut.jpg|400px]] [[File: 2013_09_09_midas_pgp1_and_alz_neurons_cut.jpg|400px]] * 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|400px]] [[File:2013_09_12_midas_alz_pcr2.jpg|400px]] ===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|400px]] [[File:2013_09_16_midas_alz_pcr2.jpg|400px]] * Total positive samples from last week = 11 [[File:2013_09_16_midas_alz_precut1.jpg|400px]] [[File:2013_09_16_midas_alz_precut2.jpg|400px]] [[File:2013_09_16_midas_alz_cut1.jpg|400px]] [[File:2013_09_16_midas_alz_cut2.jpg|400px]] ===09/17/2013=== * Extracted and processed MIDAS amplicons [[File:2013_09_17_midas_alz_pcr1.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]]
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