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Miniprep for pMP and pGL4.30
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 protocol (page 22) and a microcentrifuge.
- Cultures were selected and centrifuged (be careful to check the equilibrium of the tubes):
- pMP: 3 samples (1 to 3)
- pGL: 3 samples (1 to 3)
The usual QIAGEN protocol was followed. The R3 Invitrogen resuspension buffer was used instead of the P1 in the protocol.
- Comments
In the last centrifuge step, the cap of the pGL#2 tube got ripped off.
Then the 6 samples were checked with Nanodrop:
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.
| sample | 260/280 | 260/230 | ng/µl |
|---|---|---|---|
| pMP#1 | 1.87 | 3.11 | 175.3 |
| pMP#2 | 1.87 | 3.10 | 162 |
| pMP#3 | 1.87 | 3.14 | 170.5 |
| pGL#1 | 1.86 | 2.72 | 229.5 |
| pGL#2 | 1.86 | 2.75 | 275 |
| pGL#3 | 1.86 | 2.69 | 251 |
Transformation with pHY42 (melanopsin) for miniprep
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)
This had to be redone, since the last one (using 2 µl of DNA) didn't provide any colonies at all.
Now we used 50 µl of cells and 5 µl of DNA (at 33 ng/µl).
Meeting
Usual Monday meeting with the TAs and the professors.
