Shreffler:Notebook/Alex Daily/2012/04/04

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Sarita Buffy

  • Sarita received buffy coat from MGH (from Tues 4/3/12). I ficolled/counted (550 million PBMCs) and stripped ~400 million cells with lactic acid following Sarita's lactic acid stripping protocol. These were washed twice and resuspended in 800 µl RPMI. 400 µl were passively sensitized with serum drawn from PNOIT #965-03 (high IgE levels for Ara h 2 according to phadia results); the other 400 µl were sensitized with Sarita's own serum as a negative control for peanut-specific response.
  • Other 150 million cells used in Sarita's plasmablast staining.

TCR Sequencing

  • The transfected cells that we plated yesterday didn't grow at all. It turns out pGem chemically competent cells are ampicillin resistant, NOT kanomycin resistant. Thus, Moshe and I re-plated the cells on ampicillin plates (again, using IPTG/XGal protocol from NIU). I have a feeling that the IPTG/XGal selection may be iffy due to incomplete IPTG/XGal application on plate (using spreader beads). If the plates look bad tomorrow, we can use use ImMedia Amp Blue (provided by Mike Byrne), containing ampicillin, IPTG, and XGal in proper concentrations.
  • We plan on sending gel-purified cDNA from pcr of TCell material directly to sequence (uncloned, mix of different sequences). However, nanodrop revealed insufficient DNA concentrations, so Moshe ran a PCR to amplify the cDNA present. This was run on a standard agarose gel in confirm purity/size.


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