Novocrania cloning
Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.
08/11/14
Ran gel and set ligation.
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Bruno Vellutini
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Bruno Vellutini
Brachiopod in situ
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.
07/06/14
Added probes, embryos look good.
09/06/14
Washing day. Everything went as usual and put antibody around 18h.
10/06/14
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
11/06/14
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.
12/06/14
Stopped wnt1, wnt5 dcr1b, tdr1. Novocrania samples are looking dark ugly already…
13/06/14
Trying to clear out some of the background from fz7 and ago… letting in ethanol for many hours.
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Bruno Vellutini
Probe synthesis Ttra frizzleds Nano dlx and more
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.
09/05/14
Began probe synthesis at 10:30.
10/05/14
Done with probe synthesis, see values above.
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Bruno Vellutini
Cloning Ttra frizzled, Nano hox1, dlx, wnts
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 30/04/14
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.
01/05/14
Pic colonies for colony PCR. Run gel and set bacteria to grow.
02/05/14
Extracted minipreps and set sequencing PCR.
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Bruno Vellutini
Terebratalia in situ for FGFR, Frizzleds and others
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 14/09/13
Put probe.
16/09/13
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.
17/09/13
- Washed antibody.
- Set Cy3 TSA to double fluorescent for 1h45.
- Started developing regular NBT/BCIP at 16h.
- Stopped Fz2 around 20h, let the rest overnight.
18/09/13
- Stopped FGFR and Fz4. Let the rest continue, almost stopped Pax2/5/8, since it is almost there.
22/09/13
- Ethanol washes.
- Glycerol added.
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Bruno Vellutini
Probe synthesis Terebratalia
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 11/09/13
Everything looks alright!
12/09/13
Set transcription reaction at 10:30, stopped at 18:30.
13/09/13
Finished probe synthesis and everything went well, very good pellets. See concentration values at the table above.
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Bruno Vellutini
Brachiopod cloning
Began PCR for cloning Novocrania en, wnt1, wnt5 and Terebratalia fgfr, lfng, pax2/5/8, Fz2, Fz4, Fz5/8, Fz7, Fz10, aldh2, arm.
01/08/13
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.
02/08/13
Managed one band for engrailed, but not at the right length, slightly shorter.
03/08/13
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.
05/08/13
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 06/08/13
Sequences above dropped at the sequencing facility after miniprep extraction and sequencing pcr.
Also, set colony pcr for the genes that hadn’t worked.
07/08/13
Colony gel, putting good colonies to grow today.
08/08/13
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
