HMM is bound together with RNA; it must be kept in a RNAse-free environment in order to stay intact. Consequently, RNAse may be used to digest HMM.
HMM is acquired from Harold Smith’s lab in the Strong Memorial Hospital. They freeze drops of stock HMM in beads. The stock concentration can vary slightly, but it tends to be between 1.8 and 2.2 mg/ml. These beads are kept in the -70°C freezer in the 3rd floor equipment room, in the second cupboard from the top.
The HMM buffer (5x concentration):
- 125mM Tris pH 7.2
- 50mM MgCl2
- 50mM Imidazole
- 500mM NaCl
- 25% glycerol
Working with HMM:
1. Follow the “Preparing an RNAse-free setup” protocol.
2. Get the -70°C freezer key from the 2nd floor office.
3. Bring a bucket (for ice), a 15ml conical, and the freezer key to the 3rd floor equipment room.
4. Open the freezer and take out the 15ml conical containing the HMM beads, but leave the freezer door open.
5. Carefully shake the number of HMM beads you need into the empty 15ml conical.
The volume per bead varies with size (obviously), but is usually around 50-75uL per bead.
6. Return the HMM beads to the freezer.
7. Fill the bucket with ice. Keep the HMM on ice.
8. Use a RNAse-free micropipetter and tip to transfer the HMM into a RNAse-free centrifuge tube. This way, the volume of HMM is known.
9. Spin the HMM for 10 minutes at 13000 RPM with benchtop centrifuge.
10. Transfer all but about 5µl of the HMM sample to a new centrifuge tube. Discard the old one.
11. Prepare the desired working concentration of HMM using HMM buffer.
12. Spin down a second time. Leave this tube on ice and take care not to disturb any precipitate. The first spin only needs to be performed after a new bead is thawed, but this second spin must be performed at the start of each day.
13. At the end of the day, the HMM is kept in the refrigerator overnight. It breaks down over time, so it should not be kept for more than 1-2 days.
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