"
Contents |
Observation of the transformation from the night before
Growth on all the plates (about 20 colonies on most plates and a single colony on the control plate).
Miniprep
Team:EPF-Lausanne/Protocol/Miniprep All miniprep colonies were used to extract DNA, except SEAP1 + LS (there wasn't a lot of growth, we used the 1:2 dilution).
Nanodrop of the minipreps
Protocol: DNA Concentration Measurement
- Take a 6 µl aliquote of the DNA and put back the main DNA tube in the fridge.
- Go to the room by the E.Coli lab (LBTM, not on Friday morning!) with:
- The 6 µl aliquote
- A 10 µl pipet
- Optionally, the buffer you used for DNA elution (there might be some next to the machine).
- The machine is the NanoDrop Spectrophotometer.
- On the computer, click on "Nucleic Acid".
- Put a 2 µl drop of (nuclease-free) water on the machine's tip as you are asked to and measure.
- Clean tips (both sides) with a quarter of tissue.
- Add 2 µl of the buffer you use and click on "Blank".
- Clean tips (both sides).
- Add 2 µl of your DNA sample and click "Measure".
- Clean tips (both sides) with a tissue.
- Take 2 measurements per sample (for averaging).
- Print the report when you are done
- Click on exit.
The important numbers are:
- 260/280 ratio, must be > 1.8
- 260/230 ratio, must be > 2 (too big, > 2.5? , might mean too much salts)
- Of course the DNA concentration.
The Nanodrop gave acceptable concentrations for all of the minipreps, between 200 and 400 ng/µl.
Digestion of TNFR + pGL
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.
Protocol used for the Master mix: - 5 µl Buffer N4 10x - 5 µl BSA 10x - 1 µl XbaI (20 u/µl) - 5 µl DNA - 34 µl H20
- Comments
Re-verification of excised LovTAP & pGL fragments with a single digestion. Digestion of pGL backbone with NotI fragments generated 2922 and 1342 bp using small quantities of DNA.
Quantities in each tube:
- 5 µl buffer N2 10x - 5 µl BSA 10x - 5 µl DNA (pGL backbone) - 1µl Not1 - 34 µl H20
Quantities in each tube:
- 5µl buffer N2 10x - 5µl BSA 10x - 1µl Xba1 - 5µl DNA (excised LovTAP) - 34µl H20
Production of a new TAE solution
Protocol: TAE
To make 200 ml of %0x TAE :
- Tris base: 78.4 g
- Acetic Acid (100%): 11.4 ml
- EDTA: 20ml 0.5 M
- 292.25M --> 146.12 = 0.5 mol --> 1.46 g in 10 ml is 0.5M
- Comments
The TAE in the lab got sticky and contained unknown particles in it, so it was useful to make a new mix.
