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.
Microarray setup
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.