RNA extraction for 2nd series of Priapulus stages and L. longissimus 


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Starting an extraction for another series of Priapulus stages. Using Phase Gel Lock tubes, LPA carrier and overnight precipitation.

Embryos were at the bottom so it was a little easier to pick, but in the end I had to spin them many times to dry out the collecting tube to 100 µL. Another problem was using 1 mL for separating the phases in the gel lock tubes. They only support 750 µL. For this reason the gel was at the top in most tubes. So I collected the aqueous phase with Chema’s help and mixed an equal volume of chloroform to take the phenol out. Only after this we added the carrier (1 µL) and let it precipitate overnight.

05/05/13

Some tubes had a transparent bottom phase. After spinning this unknown droplet trapped the pellet, nothing changed after the ethanol wash. When I added 22 µL of DEPC water the pellet and droplet seem to have dissolved. In the tubes where there was a clear and nice pellet a solid case remained where the pellet was. These are the results of the NanoDrop:

Sample ID ng/ul 260/280 260/230
Pc2 oocytes 106,74 1,78 0,41
Pc2 1d 186,70 1,95 0,38
Pc2 2d 236,72 2,18 0,50
Pc2 3d 246,45 2,04 0,50
Pc2 4d 834,65 1,93 0,43
Pc2 5d 215,32 2,13 0,43
Pc2 6d 112,33 1,68 0,39
Pc2 7d 303,38 1,85 1,37
Pc2 9d 283,21 2,04 0,55
Pc2 12d 390,20 1,92 0,52
Pc2 15d 485,06 1,94 0,96
Pc2 2nd larvae 131,70 1,74 0,40
Llon mixed 288,17 1,85 0,59

Candidates for re-extraction are: oocytes, 7d, 2nd larvae