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--- | ||
title: Cell Preparation | ||
draft: false | ||
tags: | ||
- su | ||
- biology | ||
--- | ||
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## Introduction | ||
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The biological samples are introduced in the PDMS device via spotting on a glass slide. The biological samples can be: | ||
- cells, yeast (S. cerevisae) or bacterial (_E. coli_) | ||
- spores, yeast (_S. cerevisae_) or bacterial (_B. subtilis_) | ||
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This protocol descibes how to prepare the cells/spores for spotting. | ||
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## Procedure | ||
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### _S. cerevisae_ (Yeast GFP library strains) | ||
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1. Pick a glycerol stock from the strain of choice and spread on SD-His plates. The current available strains are: | ||
- [RPL8A / YHL033C](https://www.yeastgenome.org/locus/S000001025) | ||
- [RNR3 / YIL066C](https://www.yeastgenome.org/locus/S000001328) | ||
- [HUG1 / YML058W-A](https://www.yeastgenome.org/locus/S000007472) | ||
2. Incubate the plate(s) at 30°C for 18-48 hr. | ||
3. Pick one colony from the strain(s) of choice and inoculate in 4-10 ml of YPD or SD-His medium. | ||
4. Incubate for 48 hr at 30°C and 130 rpm. | ||
5. _Optional:_ Centrifuge the cells at 2400 rpm for 3 min. Resuspend the cells in 100 ul of SD-His medium. | ||
6. Place the cells in a 96-well plate for spotting. | ||
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### _E. coli_ | ||
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1. Pick a glycerol stock from the strain of choice and spread on LB plates with the appropriate antibiotic (e.g. Cam). The current available strains are _E. coli_ transformed with the following plasmids (all have Cam resistance): | ||
- [BBa_J364000](http://parts.igem.org/Part:BBa_J364000) | ||
- [BBa_J364002](http://parts.igem.org/Part:BBa_J364002) | ||
- [BBa_J364007](http://parts.igem.org/Part:BBa_J364007) | ||
2. Incubate the plate(s) at 37°C overnight. | ||
3. Pick one colony from the strain(s) of choice and inoculate in 4 ml of LB medium + antibiotic (e.g. Cam). | ||
4. Incubate overnight at 37°C and 130 rpm. | ||
5. Centrifuge the cells at 3000 rpm for 5 min. | ||
6. Resuspend the cell pellet in 100 ul LB with 10% glycerol and appropriate antibiotic (e.g. Cam). | ||
7. Place the cells in a 96-well plate for spotting. | ||
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## Spore preparation | ||
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### _S. cerevisae_ (Yeast GFP library strains mated with a query MATα strain) | ||
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1. Place well-concentrated spores (in sporulation medium) in a 96-well plate for spotting. | ||
2. _Optional:_ Centrifuge the spores at 2400 rpm for 3 min. Resuspend the cells in 100 ul of water. | ||
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## References | ||
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1. [Volpetti et al. (2017). A microfluidic biodisplay. ACS synthetic biology](https://pubs.acs.org/doi/10.1021/acssynbio.7b00088) | ||
2. [Dénervaud et al (2013). A chemostat array enables the spatio-temporal analysis of the yeast proteome. PNAS](https://www.pnas.org/doi/abs/10.1073/pnas.1308265110) |
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