Part 1: Extract DNA from selected clones (mini prep)
deciding whether to do Qiagen or homebrew kit
Qiagen: old text to revise below
- Pick up your three candidates cultures, growing in the test tubes labeled with your team colour. Label three eppendorf tubes to reflect your candidates (C1-3).
- Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube.
- Balance the tubes in the microfuge, spin them for two minutes, and remove the supernatants with the vacuum aspirator.
- Pour another 1.5 mL of culture onto the pellet, and repeat the spin step.
- Resuspend the cell pellet in 250 μL buffer P1.
- Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
- Add 250 μL of buffer P2 and mix by inversion until the suspension is a homogeneous blue colour. About 4-6 inversions of the tube should suffice.
- Buffer P2 contains sodium hydroxide for lysing.
- The blue colour comes from a special reagent that is not required for purification, but is simply used to check one's mixing technique.
- Add 350 μL buffer N3, and mix immediately by inversion until there is no blue colour (4-10 times).
- Buffer N3 contains acetic acid, which will cause the chromosomal DNA to messily precipitate; the faster you invert, the more homogeneous the precipitation will be.
- Buffer N3 also contains a chaotropic salt in preparation for the silica column purification.
- Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
- Meanwhile, prepare 3 labeled QIAprep columns, one for each candidate clone.
- Transfer the entire supernatant to the column and centrifuge for 1 min.
- Wash with 0.5 mL PB, then separately with 0.75 mL PE, with each spin step 1 min long.
- After removing the PE, spin the mostly dry column for 1 more min.
- It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
- Add 30 μL of EB to the top center of the column, wait 1 min, then spin 1 min to collect your DNA.
- EB is elution buffer, or 10 mM TrisCl (pH 8.5).
Part 2: Measure DNA concentration
Part 3: Prepare sequencing reactions
Part 4: Count colonies
Part 5: Sensitivity/specificity analysis for microsporidia primers
For next time
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