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Add SU example note
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xlxs4 committed Dec 29, 2023
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54 changes: 54 additions & 0 deletions content/Cell Preparation.md
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---
title: Cell Preparation
draft: false
tags:
- su
- biology
---

## Introduction

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_)

This protocol descibes how to prepare the cells/spores for spotting.

## Procedure

### _S. cerevisae_ (Yeast GFP library strains)

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.

### _E. coli_

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.

## Spore preparation

### _S. cerevisae_ (Yeast GFP library strains mated with a query MATα strain)

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.

## References

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)
1 change: 1 addition & 0 deletions content/index.md
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- [[Double Layer]] - PDMS chip fabrication
- [[PDMS Design]] - PDMS chip design iterations
- [[Alignment and Bonding]] - PDMS Alignment and Bonding
- [[Cell Preparation]]

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