Etchevers:Notebook/STRA6 in eye development/2008/12/08

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More trials for extractions of nuclei

Nicolas and Adeline had attempted a new trial on two E18 eyes and a preparation of kidney, to which they did multiple sonication conditions.

The eye preparation they precipitated 1/8th (0.5 mL) of the diluted sonication (in 400 uL) and got a total yield of some 2.5 ug. This compares poorly with Christelle's extraction of 50 ug from 12 C14 hearts (from which she performed 5x IPs).

I suggested we find out how much total DNA in those two eyes, so Nicolas started this with another tube:

• Tissu dans 1 ml PBS. • 50 µl proteinase K (10 mg/ml) et 120 µl 10% SDS, la nuit 45°-56ºC • (Tomorrow:) Ajouter 1,2 ml phenol, extraire, centrifuge 3000 rpm for 5 min at 4-10°C. • Pipetter surnageant a new tube, add 1.2 ml phenol, and 1.2 ml chloroform/isoamyl alcohol (24:1); centrifuge at 3000 rpm for 5 min. • Pipette the supernatant into a new tube, add 2.4 ml chloroform/isoamyl alcohol (24:1), shake by hand briefly, and centrifuge at 3000 rpm for 5 min.

Precipitate as usual (sodium acetate). I suggested using Jackie's 20 mg/ml frozen stock of proteinase K rather than the 1 year old one in the fridge from Qiagen, which I would not have recommended.

  • Heather 12:09, 8 December 2008 (EST):