Long probe for Novocrania wnt1
Set probe PCR for:
| Na wnt1 | BV387: T7 | 80 ng/µL |
| Mm pl10 | BV79: SP6 | 702 ng/µL |
and extracted.
12/05/15
Set transcription reaction and precipitation.
13/05/15
Finished probes (see values above) and made 1x dilution for wnt1.
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Bruno Vellutini
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Bruno Vellutini
Membranipora in situ and Novocrania 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.
09/05/15
Added probes just after synthesis.
11/05/15
Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.
12/05/15
Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.
13/05/15
Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.
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Bruno Vellutini
PCR mostly stem/germ cell genes of Terebratalia
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.
24/07/12
Transformation and plating of colonies.
25/07/12
PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.
27/07/12
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 -
Bruno Vellutini
Primers for more Terebratalia germline genes have arrived.
- Tt dicer1b
- Tt runx2
- Tt maelstrom
- Tt tudor1
- Tt pl10
- Tt ago
- Tt pum
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Bruno Vellutini
More Membranipora genes: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b
Set a PCR to clone: Mex3, Fez, PL10, FoxG, Syt1a, Syt1b
02/04/12
Bands with expected lengths. Purified DNA, set ligation reaction for 1h45 at RT, heatshock transformation and plated colonies with Xgal.
03/04/12
Plates were wetty, so colonies did not spread well… some were forming strings; but enough to pick. Picked white colonies for colony PCR:
09/04/12
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 -
Bruno Vellutini
Membranipora primers have arrived: Mm Syt1a, Syt1b, NK2.1, FoxG, Otp, PL10, Fez.