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We have colonies for SEAP and eGFP!
Cultures to be launched.
TNFR minipreps
10 minipreps from yesterday's cultures.
Protocol: Miniprep
The slim tubes can be centrifuged in the machine in front of the "Gel hood", at 4000 rpm for 10 min. The fatter ones, in the E. coli centrifuge by the fridge (the tip can be left inside, since it floats).
Pellets resuspended with RNase containing buffer (Resuspension Buffer R3, from Invitrogen, equivalent to Buffer P1 from Qiagen, in Sowmya's box in the fridge). Note: keep the buffer in ice if you are not bringing it back to the fridge for some minutes.
We then use the QIAGEN QIAprep Spin Miniprep Kit with their [http://www.qiagen.com/literature/render.aspx?id=370 protocol] (page 22) and a microcentrifuge.
LovTAP into pcDNA3.1
10 µl of LovTAP maxiprep (250 ng/µl) digested with HindIII and EcoRI.
10 µl of pcDNA3.1 digested with HindIII-HF and EcoRI-HF.
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
- [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp 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.
Planed:
- LovTAP to be run in gel, and purified.
- pcDNA to be run through column to take out small chuncks.
- LovTAP + pcDNA ligation
- Transform
Digest pGL with HindIII and FseI
15 µl of DNA (some 250 ng/µl) for further ligations with Fussenegger ROs.
Protocol: None
Forgot to insert protocol.
- Comments
Insert comments about what happened.
