"
Contents |
LovTAP PCR product digestion
LovTAP was digested for ligation into pcDNA3.1(+), with NotI and SpeI.
- The following mix was made
- H20: 26 µl
- N2 buffer: 5 µl
- BSA 10x: 5 µl
- NotI: 2 µl
- SpeI: 2 µl
- LovTAP DNA: 10 µl
pcDNA3.1(+)-LovTAP ligation
Protocol: Ligation
Ligation is a method of combining several DNA fragments into a single plasmid. This is often the
step following a PCR (and a PCR cleanup) or a gel extraction. You can also do a "dirty" ligation, where you follow a certain number of digestions directly by a ligation.
- Download the following spreadsheet : File:Team-EPF-Lausanne Ligation.xls
- Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
- Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
- Ligate for 2 hours at 14ºC.
- Immediately transform competent bacteria with the ligation product.
Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).
One of the digestion products for LovTAP was purified after PCR, the other one was not. We wanted to compare the efficiency of these two methods. We therefore ligated a tube of "clean" LovTAP (1:3), two tubes of "dirty" LovTAP (1:2 and 1:3) and a pGL4.30-only control (1:3).
Transformation with the ligations
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)
We used the 4 ligated tubes to transform bacteria (as usual).
