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Ligation plates observation
- There was no growth on the DH5α plates (we had plated some to test the new Amp plates we had made), negative control. This means our plates are good and our competent bacteria have not been contaminated by any Amp-containing plasmid.
- There was no growth either on the pGL4.30 ligation control that didn't have a fragment.
- There was a lot of colonies on both the pGL4.30-eGFP and pGL4.30-TNFR ligation plates (equally on "clean" and "dirty" plates, i.e. the ones that have or have not undergone a PCR cleanup). A colony PCR should be performed on these ones as soon as possible.
- Some colonies on the pMP control (without a fragment) have been observed.
- There were extremely few colonies on LovTAP-pMP, both clean and dirty. These ones should be PCR'd too.
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.
The C6 culture was miniprepped, following the usual protocol. This one is going to be sequenced to see if we can finally get pcDNA3.1(+)-LovTAP.
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.
The Nanodrop showed 270 ng/µl for this miniprep, which is reasonable.
