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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
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.
All miniprep colonies of the ligations 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.
