Probe synthesis for Terebratalia segmentation

Beginning probe synthesis for:

Tt netrin BV110 SP6
Tt patched BV115 SP6
Tt hedgehog BV129 SP6
Tt smoothened2 BV131 SP6
Tt nk2.2 BV135 SP6
Tt nk5 BV139 SP6
Mm ago2 BV25 SP6
Mm mago nashi BV27 T7
Mm germ cell less BV30 T7

Run the PCR and letting overnight to extract tomorrow.

16/05/12

Ran gel and extracted the bands. Looking ok:

18/05/12

Started probe synthesis reaction at 10:40. Precipitated around 16h.

19/05/12

Terebratalia probes had no pellet (but in fact, I don’t know if I centrifuged it). Membranipora using SP6 had a small pellet and the two T7 ones had a huge pellet. Specs are:

Gene ng/µL
Mm Ago2 293.92
Mm Magoh 3806.73
Mm GMCL 3147.70

Diluted to 50 ng/µL and stored at -20 °C.

21/05/12

Re-doing the transcription reaction for Terebratalia segmentation genes. Started at 11h, but only put at 37°C at 13h (my bad…). Precipitated at 17h30 using only 5 µL of Lithium Chloride. Could not see any pellet…

22/05/12

After centrifuging a thin pellet appeared in the bottom of the tubes. I used Low yield, but enough for making probes:

Gene ng/µL
Tt netrin 120.56
Tt patched 174.64
Tt hedgehog 120.93
Tt smo2 107.95
Tt nk2.2 211.83
Tt nk5 221.16

Diluted to 50 ng/µL and stored in the freezer.