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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.
- 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 at 37°C.
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. |
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> | We'll start off picking 12 colonies. These will end up being our 12 replicates that are passaged simultaneously day-to-day. |
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- 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.
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- 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.
- Using a sterilized, rounded pasteur pipet, pick each of the 24 colonies and spin it in a glycerol well.
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- Choose the 12 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.
- Using a sterilized, rounded pasteur pipet, pick each of the 12 colonies and spin it in a glycerol well. Store plate in the -80°C freezer.
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- With a sterile loop touch the remainder of the colony on the plate and streak onto a new MG plate, in a circular fashion, diluting as you go around.
- Let grow overnight at 37°C.
Day 3: Passaging Colonies |
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- Pick the last single colony and passage onto a new MG plate.
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- For each of the 12 plates: pick the last single colony and passage onto a new MG plate with a sterile toothpick.
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- Again, streak onto a new MG plate, in a circular fashion, diluting as you go around.
- Repeat this passaging each day.
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Freeze Clones |
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- Every 4 days, before passaging pull out the freezer plate from the -80°C freezer and, in the next row down, add 50μl 80% glycerol to each well.
- Using a sterilized, rounded pasteur pipet, pick each of the 12 colonies and spin it in a glycerol well. Return plate to the -80°C freezer.
VNTR analysis!
Note to self: Talk to Robin about designing labeled 5' primers, etc! |
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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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| 3 |
(TA)12 |
rhaT-rhaD |
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| 4 |
(A)24 |
rhaT-rhaD |
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| 5 |
(AGA)8 |
rhaT-rhaD |
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| 6 |
(GGA)8 |
rhaT-rhaD |
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| 7 |
(G)9 |
rhaT-rhaD |
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| 8 |
(G)12 |
rhaT-rhaD |
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-- Main.LindseyWolf - 18 May 2011 |
