Probe PCR for bmp/24, chordin, fgf8 and piwi1.
Extracted probe PCR gel.
Bit weird the bmp2/4, but well. Made the transcription reaction for 6:30h and put probes to precipitate.
Finish probes.
Set PCR with 60°C annealing, 1:45 elongation, 40 cycles and primers at 25mM.
| wnt1 1:10 cDNA | wnt1 1:20 cDNA |
| pax6 1:10 cDNA | pax6 1:20 cDNA |
It worked. Extracted, ligated, transformed and plated.
Colony PCR and 2 volonies were fine:
Put these to grow under the IDs:
| T7 | |
| Na wnt1 long | BV386 |
| Na wnt1 long | BV387 |
Extract miniprep and set sequencing reaction.
I selected alternate primers for getting longer fragments and improving the quality of Novocrania in situs.
Nano ptc2, wnt1, smo1, smo2, fgf8, Mmem wnt4
wnt1 did not work again, bad ptc2 again, smo1 and smo2 weakly worked, fgf8 worked, Mmem wnt4 not… Re-run Nano ptc2 with different primers:
Combination of primers 3F and 2R (395 bp fragment) was the only one that worked. Transformed:
| T7 | |
| Na wnt5 | BV352 |
| Na wnt5 | BV353 |
| Na smo2 | BV354 |
| Na fgf8 | BV355 |
| Na fgf8 | BV356 |
| Na ptc2 | BV357 |
| Na ptc2 | BV358 |
I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.
Ran gel and cut a band, but wnt1 and ptc2 did not work… OF COURSE! because fragment lengths are the following: wnt1=1239 bp, wnt5=1097 bp, ptc2=1258 bp! And the elongation time of the PCR was set to X.
Re-tried the same PCR with a longer elongation time. The result was the same. Chema suggested running nested primers to see if something appears.
Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.
Ran gel and set ligation.
I have ordered additional primers to clone more segmentation genes. Setting a PCR with 25 µM for primer concentration and 65 °C for annealing temperature.
Ttra lmx1, nk1a, nk1b, odd
So, odd failed.
Set ligation at 4 °C to go overnight (have to write…)
Set PCR.
Gel, extraction, ligation, transform.
Set colony PCR overnight.
Ran gel and put colonies to grow.
Extracted minipreps and did sequencing PCR. Left samples at the sequencing facility.
Using Andi’s primers with annealing temperature at 57 °C.
Engrailed rendered a band at the expected size (638 bp), although it is a bit spread band. It is quite possible that wnt1 had a band at the right place (718 bp), but it was very weak; I cut it anyway. Wnt5 band was also at the correct place (932 bp), but also weak.
Set ligation reaction at 16h and stored at 4°C.
Transformation and plating.
en had a lot of colonies, wnt1 had a normal amount, and wnt5 had a few colonies. I managed to do 10 colonies for the first two and 8 colonies for wnt5:
Picked colonies for minipreps
| gene | colony | T7 |
| en | 8 | BV266 |
| en | 9 | BV267 |
| en | 10 | BV268 |
| wnt1 | 1 | BV269 |
| wnt1 | 2 | BV270 |
| wnt1 | 3 | BV271 |
| wnt5 | 1 | BV272 |
| wnt5 | 2 | BV273 |
| wnt5 | 3 | BV274 |
Checked the alignment and it looks good! See files: Nano_en-wnt1-wnt5
Set PCR with 50 °C annealing temperature and 40 cycles with new primers.
Not good enough, I cut the top band of wnt1 anyway…
Did a new PCR with new primers for engrailed, wnt1, and wnt5 in Novocrania at 55 °C.
It failed…
