Questions tagged [high-throughput]

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2
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1answer
41 views

qPCR : Cross-contamination while sealing plate

I'm trying to do realtime PCR on a plasmid and I have my positive and negative controls close to each other along with a no-template control. I add 1ul of my template last into the 96 well plates (on ...
2
votes
0answers
30 views

Fisher's Method to melt p-values for CpG sites annotated to the same gene?

I have some data I am working with, and I am curious if I am able to combine p-values from a paired t-test for CpG sites in the genome using Fisher's Method to get one p-value for each unique gene. ...
0
votes
1answer
62 views

How do you obtain specific mRNA transcript levels for comparison from a Hi-Seq dataset?

I have HiSeq data from mice exposed to two conditions. I would like to answer the following question: "Is there a significant difference in mRNA transcript levels when comparing condition A to ...
0
votes
1answer
275 views

How do high-throughput/NGS sequencers calculate quality scores?

I am confused as to how quality scores are actually calculated by DNA sequencers like Illumina. For each base call, some quality predictor value is computed, based on various properties of the ...
2
votes
0answers
37 views

how to make plasma cells adhere to the bottom of a microplate?

I am isolating single plasma cells by FACS sorting into 384-well plates, with the intent to assay the supernatant and clone H/L chains from positive wells. The efficiency of the PCR is however low, ...
3
votes
1answer
118 views

Is there a photobleaching-resistant, cell-permeant viability stain in the far red part of the spectrum?

I am looking for a live-cell–impermeable dye for viability. (The cells cannot be permeabilized and fixed in this experiment.) I would prefer with excitation and emission spectra similar to Cy5, but I ...
12
votes
1answer
1k views

How to reduce edge effects in cell-based high-throughput experiments?

In high-throughput experiments where cells are cultured, treated, stained, and imaged in 384-well microplates, I frequently see significant edge effects. For instance, the following plot shows cell ...