Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.
Ran gel and set ligation.
In situ for additional Terebratalia frizzleds and repeating some Novocrania for better pictures.
| Nano | engrailed | wnt1 | wnt5 | dlx | pax258 |
| Ttra | frizzled 5/8 | frizzled 7 | dcr1b | tdr1 | ago |
Started in situ with standard procedures. 8 min protk for Novocrania and 10 min for Terebratalia.
Added probes, embryos look good.
Washing day. Everything went as usual and put antibody around 18h.
Washed with PBT several times berween 10-15 min. Then PTw 30 min washes. Started developing.
Stopped Nano dlx after 2h30min because it was done, as well as Ttra fz5/8. The rest continued developing. fz7 got pinkish really fast
fz7 and ago got really dark. I don’t know if it is background or signal, even though it looks like there is real signal within the background. I stopped them as well as engrailed and pax258, which look better than the last in situ.
Stopped wnt1, wnt5 dcr1b, tdr1. Novocrania samples are looking dark ugly already…
Trying to clear out some of the background from fz7 and ago… letting in ethanol for many hours.
| Gene | Miniprep | Antisense Enzyme | ng/µL |
| Nano dlx | BV285 | SP6 | 159 |
| Ttra fz5-8 | BV288 | SP6 | 366 |
| Ttra fz7 | BV291 | SP6 | 2439 |
| Ttra dicer1b | BV162 | SP6 | 1867 |
| Ttra tudor1 | BV167 | SP6 | 1076 |
| Ttra argonaute | BV168 | SP6 | 323 |
Started probe PCR.
Began probe synthesis at 10:30.
Done with probe synthesis, see values above.
Need to clone the remaining frizzleds from Terebratalia and wnts from Novocrania. Set overnight regular PCR with:
| Nano hox1 | Nano wnt4 | Nano wnt7 | Nano dlx | Ttra fz5/8 | Ttra fz7 |
Unfortunately hox1 and the wnts did not work. I should change something in the PCR. I extracted dlx and frizzleds and set a ligation for 4h at RT.
Transformed and plated the 3 genes and incubated at 37 °C.
Pic colonies for colony PCR. Run gel and set bacteria to grow.
Extracted minipreps and set sequencing PCR.
Starting an in situ for additional Terebratalia genes related to the segmentation project. Also a double with wnt1+wnt5.
| FGFR | Pax2/5/8 | Frizzled2 | Frizzled4 |
| Frizzled10 | LFNG | ALDH2 | Wnt1+Wnt5 |
Put probe.
Washed in situ and added anti-dig-ap 1:5000 to the first seven genes and anti-dig-pod 1:250 for the double fluorescent.
Probe PCR for Terebratalia genes (except wnt1, I had it already).
| gene | id | enzyme | utp | ng/µL |
| FGFR | BV249 | SP6 | DIG | 1618.42 |
| Pax 2/5/8 | BV251 | T7 | DIG | 1155.38 |
| Frizzled 2 | BV254 | SP6 | DIG | 1542.03 |
| Frizzled 4 | BV257 | SP6 | DIG | 1629.45 |
| Frizzled 10 | BV259 | SP6 | DIG | 978.54 |
| LFNG | BV263 | SP6 | DIG | 1137.59 |
| ALDH2 | BV265 | T7 | DIG | 1213.53 |
| WNT1 | AH23 | SP6 | DNP | 133.85 |
Everything looks alright!
Set transcription reaction at 10:30, stopped at 18:30.
Finished probe synthesis and everything went well, very good pellets. See concentration values at the table above.
Began PCR for cloning Novocrania en, wnt1, wnt5 and Terebratalia fgfr, lfng, pax2/5/8, Fz2, Fz4, Fz5/8, Fz7, Fz10, aldh2, arm.
Gel, Novocrania genes did not work. Others did, extraction and ligation.
I started a PCR for Novocrania genes using a lower annealing temperature of 55 °C.
Managed one band for engrailed, but not at the right length, slightly shorter.
I reset the ligation for: lfng, fz58 7, aldh2, arm.
Colony PCR running for the rest.
Also started a PCR with Nano wnt1 and wnt5 with annealing temperature of 50 °C.
Transformed and plated the genes that did not work previously (lfng, fz5/8, fz7, aldh2, arm).
Novocrania gel is not promising, bands are not the right size, need to design new primers.
Colony PCR went well and today I’ll put the colonies to grow.
| Animal | Gene | Colony | T7 |
| Ttra | FGFR | 1 | BV248 |
| Ttra | FGFR | 2 | BV249 |
| Ttra | FGFR | 3 | BV250 |
| Ttra | Pax 2/5/8 | 1 | BV251 |
| Ttra | Pax 2/5/8 | 2 | BV252 |
| Ttra | Pax 2/5/8 | 3 | BV253 |
| Ttra | Fz2 | 1 | BV254 |
| Ttra | Fz2 | 2 | BV255 |
| Ttra | Fz2 | 3 | BV256 |
| Ttra | Fz4 | 2 | BV257 |
| Ttra | Fz4 | 3 | BV258 |
| Ttra | Fz10 | 2 | BV259 |
| Ttra | Fz10 | 3 | BV260 |
| Ttra | Fz10 | 4 | BV261 |
| Nano | En | 3 | BV262 |
Sequences above dropped at the sequencing facility after miniprep extraction and sequencing pcr.
Also, set colony pcr for the genes that hadn’t worked.
Colony gel, putting good colonies to grow today.
Extracted minipreps for Lfng 3 and 5 and Aldh2 1. Sequencing PCR and dropped samples in the facility.
| Gene | ID |
| LFNG c3 | BV263 |
| LFNG c5 | BV264 |
| ALDH2 c1 | BV265 |