Team:Danci-K8/Experiments

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Protocols

Taqman assay

SNPs are screened using the Fluidigm BioMark apparatus (Fluidigm Corp.) with 16 Assay- specific TaqMan. Assays along with PCR master mix run by loading 5 microliter into each well of the primed 96.96 Fluidigm Chip. The chip is then placed in the integrated fluidic circuit controller and loaded before analysis with the BioMark reader. The following thermal cycling protocol are usually used: 50˚C (2 minutes), 70˚C (30 minutes), 25˚C (10 min), 50˚C (2 minutes), and 95˚C (4 minutes). This is followed by 40 cycles of 95˚C (10 seconds) and 61˚C (30 seconds).

Data is analysed and cycle threshold (CT) values are determined using BioMark PCR analysis software (Fluidigm Corp.), and automated genotyping calling is carried out using an algorithm based on the change in CT (DCT) values between wild-type and mutant calling.

Taste SNPs will be marked by FAM dye, Non-taste SNPs will be marked by VIC dye.

In vitro cutting of dsDNA using Cas9/synthetic RNA

The protocol is as follows (though may need some calibration): In order to test in vitro, the RNAs you ordered will have to include a 20nt spacer that matches to the DNA you wish to cut in vitro (which could be a plasmid, dsDNA/PCR product etc.) If you are using plasmid DNA, it is sometimes better to linearize the plasmid first so you can see different fragment sizes on a gel. Alternatively, you can visualize cut plasmid on a gel vs. supercoiled.

  1. Be sure to work in a RNAse free environment. Wipe surfaces down with RNAse Zap if possible.
  2. Resuspend your crRNA and tracrRNA. They are 2nmol, so add 20ul of 1X TE Buffer to create 100uM (100pmol/ul) stocks. For your working dilution, dilute these 100uM stocks to 10uM by adding 2ul of your working stock to 18ul of 1X TE buffer, to give you 20ul of 10uM (10pmol/ul). (TE buffer not provided)
  3. To anneal the crRNA and tracrRNA, use 4ul of each 10uM RNA (cr/tracr) + 32ul of annealing Buffer (provided) for a total of 40ul. This will make your final concentration 1uM (1pmol/ul). To anneal, heat to 78 °C for 10 minutes on a heating block or thermocycler block, then at 37 °C for 30 minutes. Then, take it out and leave it on the benchtop to cool down (5 minutes is usually sufficient).
  4. Normalize the concentration of the DNA you wish to cut to 50ng/ul.
  5. Set up the Cas9/guide RNA cutting assay as follows: (1x reaction)
    • 2ul of 1uM guide RNA (annealed crRNA/tracrRNA)
    • 2ul of 1uM Cas9 nuclease
    • 4ul of DNA (50ng/ul)
    • 2ul of 10x buffer. We use NEB 3.1 buffer (not provided)
    • 10ul of nuclease-free water
    Heat at 30C for 45 mins, then 65C for 15 mins. PCR block works great for this.
  6. To visualize on a gel, load 10ul of this digest with loading dye.

Notes:

  1. A ratio of 1:1 for crRNA and tracrRNA works fine for this assay. However, we have recently observed that a 2:1 ratio (crRNA:tracrRNA) provides slightly better annealing.
  2. As per the new protocol (attached), we recommend using a TE buffer that is around pH7.4. If you have something between pH7-8, it should be fine.

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