Set probe PCR for:
| Na wnt1 | BV387: T7 | 80 ng/µL |
| Mm pl10 | BV79: SP6 | 702 ng/µL |
and extracted.
Set transcription reaction and precipitation.
Finished probes (see values above) and made 1x dilution for wnt1.
Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.
| wnt1 | dlx | bmp2/4 | chordin | fgf |
| nanos | vasa | piwi1 | piwi2 | pl10 |
| Na wnt1 |
Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.
Added probes just after synthesis.
Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.
Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.
Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.
Setting up a pcr with gradient (63 +- 3 °C) at 12h:
| Gene | Primers Tm mean |
| Mm Six3/6 | 64.10 |
| Tt Dicer1b | 61.60 |
| Tt Runx | 63.15 |
| Tt Mael | 65.15 |
| Tt Piwia | 65.55 |
| Tt Tudor1 | 61.95 |
| Tt Ago | 62.95 |
| Tt PL10 | 63.10 |
| Tt Pum | 65.35 |
Extraction and ligation overnight at 4°C, except for MAEL.
Transformation and plating of colonies.
PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.
Put positive colonies to grow for minipreps.
| BV162 | Terebratalia transversa | Dicer1b |
| BV163 | Terebratalia transversa | Dicer1b |
| BV164 | Terebratalia transversa | Runx2 |
| BV165 | Terebratalia transversa | Runx2 |
| BV166 | Terebratalia transversa | Runx2 |
| BV167 | Terebratalia transversa | Tudor1 |
| BV168 | Terebratalia transversa | Argonaute |
| BV169 | Terebratalia transversa | Argonaute |
| BV170 | Terebratalia transversa | Argonaute |
| BV171 | Terebratalia transversa | NK6 |
| BV172 | Terebratalia transversa | NK6 |
| BV173 | Terebratalia transversa | NK6 |
| BV174 | Terebratalia transversa | Wnt7 |
| BV175 | Terebratalia transversa | Wnt7 |
| BV176 | Terebratalia transversa | Wnt7 |
Primers for more Terebratalia germline genes have arrived.
Set a PCR to clone: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b
Bands with expected lengths. Purified DNA, set ligation reaction for 1h45 at RT, heatshock transformation and plated colonies with Xgal.
Plates were wetty, so colonies did not spread well… some were forming strings; but enough to pick. Picked white colonies for colony PCR:
Sequencing PCR and dropped in the SeqLab.
| BV75 | Membranipora membranacea | Mex3 | T7 |
| BV76 | Membranipora membranacea | Fez | T7 |
| BV77 | Membranipora membranacea | Fez | T7 |
| BV78 | Membranipora membranacea | Fez | T7 |
| BV79 | Membranipora membranacea | PL10 | T7 |
| BV80 | Membranipora membranacea | PL10 | T7 |
| BV81 | Membranipora membranacea | PL10 | T7 |
| BV82 | Membranipora membranacea | FoxG | T7 |
| BV83 | Membranipora membranacea | FoxG | T7 |
| BV84 | Membranipora membranacea | FoxG | T7 |
| BV85 | Membranipora membranacea | Syt1a | T7 |
| BV86 | Membranipora membranacea | Syt1a | T7 |
| BV87 | Membranipora membranacea | Syt1a | T7 |
| BV88 | Membranipora membranacea | Syt1b | T7 |
| BV89 | Membranipora membranacea | Syt1b | T7 |
| BV90 | Membranipora membranacea | Syt1b | T7 |
Membranipora primers have arrived: Mm Syt1a, Syt1b, NK2.1, FoxG, Otp, PL10, Fez.