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Further Checks on LovTAP in pMP
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- NEBcutter for finding cutting enzymes.
- Double Digest Finder for the parameters.
- Calculate the amounts required of:
- DNA
- Buffer (usually from 10x to 1x)
- BSA, if needed (usually from 100x to 1x)
- Enzymes (depends on the amount of DNA)
- Water
- Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
- Mix all the ingredients, except DNA, in a tube.
- Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
- Distribute the mix in as many tubes as DNA samples and add the DNA.
- Keep in the Thermomixer at the recommended temperature.
Sowmya's recommended amounts (50 µl total solution):
- 5 µl of 10x buffer
- 0.5 µl of 100x BSA
- 1 µl of each enzyme
- 5 µl of DNA
- 37.5 (up to 50 µl) of water.
Protocol based on what was done on July the 4th.
Digestion of L9, L5 (LovTAP) with EcoRI and HindIII, and also with NotI and SpeI.
Digestion of the eGFP PCR product (GC, HF) with EcoRI and HindIII.
Digestion of the eGFP PCR product (GC, HF) with NotI and SpeI.
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).
Ligation of pGL with GFP at a ratio of 1:3 respectively.
Ligation of pGL with TNFR at a ratio of 1:3.
Ligation of cut pGL alone for control (also 1:3).
Ligation of pMP with LovTAP at a ratio of 1:3.
Ligation of pMP for control.
Ligation of pcDNA3.1(+) with LovTAP at a ratio of 1:3.
Ligation of cut pcDNA3.1(+) alone for control (1:3).
Transformation of the ligation products
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)
Bacteria were transformed and plated.
