Arichard:Notebook/2012/September
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September, 2012[edit]
12th[edit]
- Today I learned how to create links and pages on the lab wiki
13th[edit]
We received 4 new samples today, all nuclei from Alzheimer's patient: CTX+ (Neuronal cortex), CTX- (Non-neuronal cortex), CBL+ (Neuronal cerebellum), and CBL- (Neuronal cerebellum).
- 2401 CTX+ needs no dilution.
- 2401 CBL+ needs 1:10 dilution.
- Jeff didn't say what dilutions are needed for 2401 CTX- and 2401 CBL-.
- Other samples are as follows
- Two types of sea bacteria: Synechococcus and prochlorococcus. These samples are already at the right concentrations, 10-20 parts/ul.
- Skin bacteria sample from microbiome project. Heterogeneous. I need to ask Jeff about these dilutions.
- Control brain nuclei: 60831 FITC+ (Neuronal) and 60831 FITC- (Non-neuronal). These samples need to be diluted 1:10 in PBS (e.g., 5 ul:45 ul).
- Cell-free, metaphase chromosomes: GM20431 at 150 parts/ul. Optimal dilution for this sample has not been determined, so we are currently doing both 1:1 (meaning no dilution) and 1:5, both 50 ul total volume.
- Shadowed Jeff, 2nd time watching MDA. Sample setup below. Non-neuronal Alzheimer's samples not included today.
Used | Used | Used | Skin bac | Chrom 1:1 | Ctl ctx neuron | Alz ctx neuron | Unused |
Used | Used | Used | Sea bac | Chrom 1:5 | Ctl ctx non | Alz cbl neuron | Unused |
- Pulled, bent, and sigmacoted 18 glass micropipettes.
- Will start MDA on my own on Monday while Jeff is away for Rosh Hashanah. Same samples as today.
- Will also start CEL-Seq either Monday or Tuesday. Probably Monday since Tuesday will be busier with MDA amplicon extraction
14th[edit]
- MDA amplicon extraction
- The skin and the sea bacteria samples looked good. Extracted 3 wells from each.
- Only identified 2 positive wells for the 1:1 chromosome dilution.
- 8 samples: 3 skin, 3 sea, 2 1:1 chrom.
- Processed through to size selection. Samples stored at 4 degC.
- Jeff uses Nextera barcodes 25-48. Sam used 49-72 and these may become mine.
- Will do light sequencing (low coverage, 0.001X) to confirm quality and assay for contamination before actual sequencing run.
- Alan will do a paired-end run early next week.
17th[edit]
- Started MDA on samples: Skin, sea, chrom 1:1, chrom 1:5, ctl. neuron., ctl non-neuron., alz. ctx. neuron., alz. cbl. neuron..
- Live image on GFP channel (200 ms exposure) did not look quite right when I started the experiment. The curtain was closed and the laser was on. I'll know tomorrow whether or not it worked.
18th[edit]
- MDA worked fine. There seems to be a pretty fair amount of variability in background intensity on the GFP channel. It also varies between arrays.