Oct 05, 2021
Agarose gel pads for live Ashbya imaging
This protocol is a draft, published without a DOI.
- ameya.jalihal 1
- 1UNC Chapel Hill
- Gladfelter Lab
Protocol Citation: ameya.jalihal 2021. Agarose gel pads for live Ashbya imaging. protocols.io https://protocols.io/view/agarose-gel-pads-for-live-ashbya-imaging-bys5pwg6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 05, 2021
Last Modified: October 05, 2021
Protocol Integer ID: 53821
Keywords: agarose gel pads for live ashbya imaging, agarose gel pad, live ashbya imaging, agarose gel, imaging, gel, live ashbya
Abstract
A protocol to prepare agarose gel pads for live Ashbya imaging.
Materials
2X LFM
autoclaved dd H2O
Overnight Ashbya culture
Depression slide
Agarose
Large beaker for water bath
2 50 mL conicals
1 15 mL conical
Troubleshooting
Safety warnings
Agarose is hot. Use gloves when handling the beaker and the tube.
Before start
Rinse a depression slide with ethanol and have coverslips on hands.
Resuspension of Ashbya
10m
Note
- Make 20 mL 1X LFM stock before you start
- Always check LFM tube for contamination
- Reconstitute with ddH2O
Spin down overnight Ashbya culture to pellet cells in 15 mL conical tubes 00:05:00 300 RPM
5m
Resuspend in 4 mL 1X LFM. Spin down again. 00:05:00 300 RPM
5m
Resuspend in 1 mL 1X LFM. Incubate on the rotator at 30 °C until ready to make gel pad, or for 00:30:00 .
30m
Making agarose pads
Note
Depression slides live on Grace's desk. These are expensive, and we reuse them. Don't throw these out! Wash thoroughly with water and ethanol, dry them, and replace them in the box.
Aliquot 10 mL of 1X LFM from step 1 into a 50 mL conical.
Measure 200 mg agarose.
Fill a 500 mL beaker with water to make a water bath and microwave the tube in 00:00:30 increments until the agarose is dissolved. Shake the tube to dissolve agarose.
30s
Pipet 200 µL of the agarose solution into the depression on a depression slide.
Drag a coverslip across the depression to create the pad. After about 2-5 min, gently lift the coverslip off. The gel pad is now ready to use.
When ready, add 50 µL of the cells to the center of the pad. Gently cover it with a coverslip, and press with a kimwipe to squish the cells. Cells are now ready to image.
Imaging