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Miniprep cultures from the transformed colonies, DNA concentration measurement
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.
Since there was nothing in the pHY42 plates, we cheched the concentration of the DNA we had extracted from the paper with a Nanodrop (average over 2 samples, both very similar):
- 33.4 ng/µl of DNA
- 260/230 : 0.33
- 260/280 : 0.87
There was not much DNA, and the curves seemed extremely contaminated. From this, we concluded we had to be extremely careful with the DNA from paper, because it was clearly not very clean and not very concentrated.
Protocol: Prepare Plasmid Extraction (culture for Miniprep)
- Select and number colonies on the plates.
- Prepare tubes of LB medium with the correct quantity of antibiotics (100 µg/ml for Amp, Spc or chloramphenicol).
- Amp can be found in the -20ºC freezer at Ecoli, labeled as "stock". It is 100 µg/µl, or 1000x.
- The tubes to be used are the 14 ml round bottom found in front of the iGEM drawers (Falcon). Culture with cap in the first step (loose) and close to the second step after culture.
- Touch each colony with a clean pipette tip and put it in a tube.
- Put in incubator.
We went ahead with the other 2 plasmids:
- Added 40 µl of Amp (100 µg/µl) to 40 ml of LB: 4000 µg / 40 ml = 100 µg/ml
- Distributed in 6 Falcon tubes, 5 ml each.
- Picked 3 colonies from each plate, pMP and pGL.
- Left overnight (starting at 19:30) at 37ºC.
