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| META TOPICPARENT |
name="ProtocolList" |
Day 1: Plating the Mixed Population
- Find microsatellite-containing strains in the -80°C freezer. They will be labeled with the appropriate microsatellite sequence located in the rhamnose operon (eg. "(CA)12"). See below for the list of microsatellites.
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- Using careful sterile technique, dilute 1:1000 in saline by adding 10μl frozen stock in 10ml saline.
- Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline.
- Plate 100μl on minimal glucose (MG) plates. Label plates with strain and date.
- Let grow overnight.
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Day 2: Picking Colonies |
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> | We'll start off picking 24 colonies. These will end up being our 24 replicates that are passaged simultaneously day-to-day.
- Get out a sterile 384 well freezer plate. Label with strain and date. In the top row, add 50μl 80% glycerol. Sterility is very important here.
- Choose the 24 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.
Freeze "Ancestral" Clones |
| | Day 3: Passaging Colonies |
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> | Freeze Clones |
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| Number |
Sequence |
Position |
Fluorescent Tag? |
| 1 |
(CA)6 |
rhaT-rhaD |
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| 2 |
(CA)12 |
rhaT-rhaD |
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