Christopher Anderson Notebook
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==[[User:JCAnderson|JCAnderson]] 21:32, 11 September 2007 (EDT)== | ==[[User:JCAnderson|JCAnderson]] 21:32, 11 September 2007 (EDT)== | ||
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I did 1mL cultures in LB+100ug/mL ara of the pir controllers of pBACr-AraGFP with and without 5mM iron. I grew for 5 hrs at 37 in a block, pelleted, then did cytometry. Red is no iron, green is with iron. Looks mighty nice! I've decided to examine 2, 6, and 7 further, so I mini'd, diluted 10x, and transformed lefty cells to sep. | I did 1mL cultures in LB+100ug/mL ara of the pir controllers of pBACr-AraGFP with and without 5mM iron. I grew for 5 hrs at 37 in a block, pelleted, then did cytometry. Red is no iron, green is with iron. Looks mighty nice! I've decided to examine 2, 6, and 7 further, so I mini'd, diluted 10x, and transformed lefty cells to sep. | ||
Latest revision as of 01:37, 12 September 2007
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JCAnderson 21:32, 11 September 2007 (EDT)
I did 1mL cultures in LB+100ug/mL ara of the pir controllers of pBACr-AraGFP with and without 5mM iron. I grew for 5 hrs at 37 in a block, pelleted, then did cytometry. Red is no iron, green is with iron. Looks mighty nice! I've decided to examine 2, 6, and 7 further, so I mini'd, diluted 10x, and transformed lefty cells to sep.
JCAnderson 11:56, 9 September 2007 (EDT)
Alright, that took a while. What ended up working was taking the yfbE-pir composite part (no rbs), dropping that in the pSC101 plasmid, then doing EIPCR with dt013/14, and transforming a whole lot of material by electroporation. I only had about 50 members to the library in the end, but there were plenty of hits in there. I'm not sure how many, because transforming the lib with pBACr-AraGFP came out severely contaminated. I was able to find green colonies, though, and picked 8 of them, plating with and without iron (and Ara to turn on GFP). Several of those look nice and tight.
JCAnderson 21:35, 20 August 2007 (EDT)
I'm going to finish off the iron-pir construct David started. I set up a DT013/DT014 EIPCR on I716119 Clone 1 with 4K55 Expand. I716119 is the promoter-pir construct with no rbs in pBca9145. The oligo will put in an rbs library. i'll sub with SpeI/DpnI tomorrow.
