---++ Day 1: Plating the Mixed Population 1. 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)<sub>12</sub>"). See below for the list of microsatellites. 1. Using _careful_ sterile technique, dilute 1:1000 in saline by adding 10μl frozen stock in 10ml saline. 1. Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline. 1. Plate 100μl on minimal glucose (MG) plates. Label plates with strain and date. 1. Let grow overnight at 37°C. ---++ Day 2: Picking Colonies We'll start off picking 12 colonies. These will end up being our 12 replicates that are passaged simultaneously day-to-day. 1. 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._ 1. 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. 1. 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. 1. 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. 1. Let grow overnight at 37°C. ---++ Day 3: Passaging Colonies 1. For each of the 12 plates: pick the last single colony and passage onto a new MG plate with a sterile toothpick. 1. Again, streak onto a new MG plate, in a circular fashion, diluting as you go around. 1. Repeat this passaging each day. ---++ Freeze Clones 1. 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. 1. 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!* | *Number* | *Sequence* | *Position* | *Fluorescent Tag?* | | 1 | (CA)<sub>6</sub> | _rhaT-rhaD_ | | | 2 | (CA)<sub>12</sub> | _rhaT-rhaD_ | | | 3 | (TA)<sub>12</sub> | _rhaT-rhaD_ | | | 4 | (A)<sub>24</sub> | _rhaT-rhaD_ | | | 5 | (AGA)<sub>8</sub> | _rhaT-rhaD_ | | | 6 | (GGA)<sub>8</sub> | _rhaT-rhaD_ | | | 7 | (G)<sub>9</sub> | _rhaT-rhaD_ | | | 8 | (G)<sub>12</sub> | _rhaT-rhaD_ | | -- Main.LindseyWolf - 18 May 2011
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Topic revision: r4 - 2011-05-19 - LindseyWolf