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By the end of the workshop, you will have a clear picture of how this powerful technology works, and what it looks like in practice. | By the end of the workshop, you will have a clear picture of how this powerful technology works, and what it looks like in practice. | ||
Lisa Thalheim is a Staff Security Engineer at Apple. These days, Lisa spends her time going back and forth between finding novel or neglected security issues on the one hand, and designing and driving systemic solutions for them on the other - be that by getting involved in systems design, creating engineering guidance and improved processes, or writing better tools. | |||
=== Protocols === | |||
Notes before starting | |||
* Perform all centrifugation steps at room lemperature [15-25°C). | |||
* lf necessary, redissolve any precipilates in Buffer API and Buffer AW I cond | |||
* Add ethanol to Buffer AW1 and Buffer AW2 concentrates. | |||
* Preheat a water bath or healing block to 65°C. | |||
# Disrupt samples (s100 mg wel weight or ≤20 mg lyophilized tissue) using the | |||
# TissueRuptor*, the Tissuelyser Il or a mortar and pestle. | |||
# Add 400 pl Buffer API and 4 ul RNase A. Vortex and incubale for 10 min al 65 Invert the lube 2-3 limes during incubation. Note: Do not mix Buffer API and RNase A before use. | |||
# Add 130 pl Buffer P3. Mix and incubate for 5 min on ice. | |||
# Recommended: Centrifuge the lysate for 5 min at 20,000 × g (14,000 rpm). | |||
# Pipel the lysate into a GlAshredder spin column ploced in a 2 ml collection lube | |||
# Transfer the Rowshrough into a new tube without disturbing the pellet if present. 1.5 volumes of Buffer AW/I, and mix by pipetting. | |||
# Transfer 650 ul of the mixture into a DNeasy Mini spin column placed in a 2 ml collection lube. Centrifuge for I min at 26000 x g (8000 rpm). Discard the flor through. Repeat this step with the remoining sample. | |||
# Place the spin column into a new 2 ml collection tube. Add 500 ul Buffer AW2, centrifuge for 1 min at 26000 x g. Discard the flow through. | |||
# Add another 500 pl Buffer AW2. Centrifuge for 2 min at 20,000 x g. Note: Remove the spin column from the collection lube carefully so that the col not come into contact with the Rowthrough. | |||
# Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube. | |||
# Add 100 pl Buffer AE for elution. Incubate for 5 min al room Centrifuge for 1 min at 26000 x g. | |||
# Repeat step 11. | |||
Latest revision as of 13:23, 18 October 2023
With Lisa Thalheim
20.05.2023, 21.05.2023
This workshop explores the cutting-edge technology of DNA sequencing. The instructors will guide you through the basics of the technology, as well as a live DNA sequencing demonstration, all the way from sample preparation to data analysis.
By the end of the workshop, you will have a clear picture of how this powerful technology works, and what it looks like in practice.
Lisa Thalheim is a Staff Security Engineer at Apple. These days, Lisa spends her time going back and forth between finding novel or neglected security issues on the one hand, and designing and driving systemic solutions for them on the other - be that by getting involved in systems design, creating engineering guidance and improved processes, or writing better tools.
Protocols
Notes before starting
- Perform all centrifugation steps at room lemperature [15-25°C).
- lf necessary, redissolve any precipilates in Buffer API and Buffer AW I cond
- Add ethanol to Buffer AW1 and Buffer AW2 concentrates.
- Preheat a water bath or healing block to 65°C.
- Disrupt samples (s100 mg wel weight or ≤20 mg lyophilized tissue) using the
- TissueRuptor*, the Tissuelyser Il or a mortar and pestle.
- Add 400 pl Buffer API and 4 ul RNase A. Vortex and incubale for 10 min al 65 Invert the lube 2-3 limes during incubation. Note: Do not mix Buffer API and RNase A before use.
- Add 130 pl Buffer P3. Mix and incubate for 5 min on ice.
- Recommended: Centrifuge the lysate for 5 min at 20,000 × g (14,000 rpm).
- Pipel the lysate into a GlAshredder spin column ploced in a 2 ml collection lube
- Transfer the Rowshrough into a new tube without disturbing the pellet if present. 1.5 volumes of Buffer AW/I, and mix by pipetting.
- Transfer 650 ul of the mixture into a DNeasy Mini spin column placed in a 2 ml collection lube. Centrifuge for I min at 26000 x g (8000 rpm). Discard the flor through. Repeat this step with the remoining sample.
- Place the spin column into a new 2 ml collection tube. Add 500 ul Buffer AW2, centrifuge for 1 min at 26000 x g. Discard the flow through.
- Add another 500 pl Buffer AW2. Centrifuge for 2 min at 20,000 x g. Note: Remove the spin column from the collection lube carefully so that the col not come into contact with the Rowthrough.
- Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube.
- Add 100 pl Buffer AE for elution. Incubate for 5 min al room Centrifuge for 1 min at 26000 x g.
- Repeat step 11.