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Bruno Vellutini
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Bruno Vellutini
Novocrania wnt1 one more time
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 07/05/15
It worked. Extracted, ligated, transformed and plated.
08/05/15
Colony PCR and 2 volonies were fine:
Put these to grow under the IDs:
T7 Na wnt1 long BV386 Na wnt1 long BV387 09/05/15
Extract miniprep and set sequencing reaction.
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Bruno Vellutini
Cloning more segmentation genes
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 -
Bruno Vellutini
Cloning Novocrania patched and longer clones of wnt1, wnt5
I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.
26/11/14
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.
28/11/14
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.
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Bruno Vellutini
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Bruno Vellutini
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Bruno Vellutini
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Bruno Vellutini
Novocrania one more time
Using Andi’s primers with annealing temperature at 57 °C.
11/09/13
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.
12/09/13
Transformation and plating.
13/09/13
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:
16/09/13
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 17/09/13
- Extracted plamids.
18/09/13
- Set sequencing pcr at 10:30.
- Dropped sequences at the facility at 14:30.
22/09/13
Checked the alignment and it looks good! See files: Nano_en-wnt1-wnt5
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Bruno Vellutini
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Bruno Vellutini