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='''VA-hospital sorting on Alex-FISH E.coli (EtOH-fixed) - 30min-hybridization samples'''= ==Background== *The new FISH-labeled E.coli was performed with only '''30min hybridization''' (Made on 06-07-'10) *For this sorting, I pre-run a 1000cell sorting to cover full plate before the real sample sorting ==Procedure== *Add 900uL 1XPBS into the sample (100uL) in the amber tube. *Material need for sorting: **8-well PCR tube stirps (on 96-well rack). Filled up 3uL 1X PBS in PCR tubes. UV-treatment for 15min. **UV clean sealing film (Bio-rad) **UV clean zip bag for transporting sample + rack Real sorting arrangement 1 2 3 4 5 6 7 8 9 10 11 12 - Strip# (Strips are in vertical arrangement) ------------------------------------------------- H 0 0 0 0 0 0 0 0 0 0 0 0 G 1 1 1 1 1 1 1 1 1 1 1 1 F 1 1 1 1 1 1 1 1 1 1 1 1 E 1 1 1 1 1 1 1 1 1 1 1 1 D 1 1 1 1 1 1 1 1 1 1 1 1 C 1 1 1 1 1 1 1 1 1 1 1 1 B 10 10 10 10 10 10 10 10 10 10 10 10 A 100 100 100 100 100 100 100 100 100 100 100 100 -------------------------------------------------- ==Results== [[File:sam060910-Ecoli sorting-new.jpg|800px]] ==Discussion== *The 30min hybridization showed similar results as 90min hybridization protocol. *'''There still have two peaks showing when the sorting number is increased'''. *The background is very low in this result compared to the previous results (03-30-'10). '''I am not sure if the short-hybridization helps to reduce the negative-labeling'''. **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-30
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