20.109(S13):DNA sequencing and primer analysis (Day5)

Introduction

miniprep

sequencing

Protocols

Part 1: Extract DNA from selected clones (mini prep)

deciding whether to do Qiagen or homebrew kit Qiagen: old text to revise below

1. 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).
2. Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube.
3. Balance the tubes in the microfuge, spin them for two minutes, and remove the supernatants with the vacuum aspirator.
4. Pour another 1.5 mL of culture onto the pellet, and repeat the spin step.
5. Resuspend the cell pellet in 250 μL buffer P1.
   • Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
6. 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.
7. 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.
8. 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.
9. Transfer the entire supernatant to the column and centrifuge for 1 min.
10. Wash with 0.5 mL PB, then separately with 0.75 mL PE, with each spin step 1 min long.
11. 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.
12. 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

Reagent list

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