Mar 09, 2022

Public workspaceAgarose gel pads for live Ashbya imaging

This protocol is a draft, published without a DOI.
  • 1UNC Chapel Hill
avatar.png
Icon indicating open access to content
QR code linking to this content
Protocol Citation: ameya.jalihal, Grace 2022. Agarose gel pads for live Ashbya imaging. protocols.io https://protocols.io/view/agarose-gel-pads-for-live-ashbya-imaging-bythpwj6
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: Working
We use this protocol and it’s working
Created: October 05, 2021
Last Modified: March 09, 2022
Protocol Integer ID: 53833
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
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
Resuspension of Ashbya
10m
10m

Note

  • Make 20 mL 1X LFM stock before you start
  • Always check LFM tube for contamination
  • Reconstitute with ddH2O


Critical
Spin down overnight Ashbya culture to pellet cells in 15 mL conical tubes Duration00:05:00 300 RPM



5m
Centrifigation
Use a pipette to remove as much of the AFM as you can while leaving the cells at the bottom. Resuspend in 4 mL 1X LFM. Spin down again. Duration00:05:00 300 RPM. Either pipette pelleted cells from here onto the finished gel pad (steps 2.1-2.6), or do step 1.3 if the cells are too dense.
5m
Centrifigation
Wash
Optional: Resuspend in 1 mL 1X LFM. Incubate on the rotator at Temperature30 °C until ready to make gel pad, or for Duration00:30:00 .
30m
Incubation
Wash
Making agarose pads
Making agarose pads
30s
30s
depression slide
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.


Critical
Aliquot Amount10 mL of 1X LFM from step 1 into a 50 mL conical.
Pipetting
Measure Amount200 mg agarose.
Fill a 500 mL beaker with water to make a water bath and microwave the tube in Duration00:00:30 increments until the agarose is dissolved. Shake the tube to dissolve agarose.

30s
Pipet Amount400 µL of the agarose solution into the depression on a depression slide.
Pipetting
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.




steps 2.1-2.5
When ready, add Amount50 µL of the cells to the center of the pad. Gently cover it with a coverslip, and press gently with a kimwipe to squish the cells.
Pipetting
Stick a glass Pasteur pipette into a beaker of valap (you want to get the wax into the pipette). Then hold this over a Bunsen burner until the wax melts, and then apply around the edges of the coverslip to seal it:


The slide is now ready to image.