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Contents |
Purification of Biobricked LovTap, SEAP and TNFR
pcr purification kit from machery nigel was used.
Restriction digest of pCDNA3 (-), Lovtap, SEAP and TNFR
Team:EPF-Lausanne/Protocol/RestrctionDigest
SEAP and TNFR were cut w/ EcoRI and PstI
LovTap was digested with NheI and KpnI
PcDNA3(-) was cut with NheI and KpnI
Ligations
LovTap was ligated with pcDNA3(-)
SEAP was ligated with PGL
Transformations
To create a stock of useable plasmid for further cloning we transformed with
- PCEP4
- pcDNA3.1(-)
One more readout for the melanopsin channel experiments was transformed.
PGL-SEAP
And so was our biobricked lovtap.
PcDNA3(-) - Lovtap
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
- Comments
Insert comments about what happened.
