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=FISH with "Alexa 488 EBU 338 9/03/10 probe" on ETOH E. coli and ETOH/RLV MB= ==Background== *Previously, we tested PU on E. coli and MB on 10/13 but only could get the universal probe to work on E. coli -> wanted to move forward to try to get probe to work on MB *This time, we tested a new MB template made on 10/14, and also changed the dilution from 1/2 to 1/5 *Probe: "Alexa 488 EBU 338 9/03/10" *Template: ETOH E. coli (7/23) -> new E. coli cultured sample ETOH MB (10/14) RLV MB (10/14) *Rxn's: 1=ETOH E. coli PU 2=ETOH E. coli PN 3=ETOH MB PU 4=ETOH MB PN 5=RLV MB PU 6=RLV MB PN ==Procedure== *Prepared 2M NaCl, 0.1% SDS, 2M Tris (was stored in the 1.5ml tube that was previously tested for correct pH), 0.5 EDTA, and PBS solutions (used no UV/filter PBS because not sorting) **Vortex each reagent before use *Thaw the pre-fixed bacteria E. coli/MB samples (stored in -80C) **Used ETOH E. coli (7/23) -> no dilution because already decreased template by half during fixation **Used ETOH/RLV-fixed MB (10/14/2010) -> 1/5 dilution for MB *Pellet the samples by '''10,000xg, 5min at RT'''. Remove the supernatant. *Wash palettes with 500ul 1xPBS twice (w/ 10,000xg, 5min centrifuge after each wash) *After last washing, re-centrifuge the pellet again to remove the supernatant completely '''(10,000xg, 1min)'''. *Pre-warm the incubator to 46C '''Prepare H-buffer in foil wraped, clear low-binding 1.5-ml tube''' (didn't prepare H-buffer until every washing was done) Because same probe, prepared H-buffer for each probe rxn's separately 1 rxn --------------------------------------- H2O 86 = used nuclease-free water 2M NaCl 90 0.1% SDS 20 2M Tris 2 Probe(1000ng/uL) 2 -------------------------------------- 200 repeat pipette mix 5x *Resuspend the pellet (in 1.5mL tubes) with 200uL H-buffer. Make sure to break up pellet thoroughly for complete hybridization. *Vortex each sample for 5sec *Incubate at mixture at 46C for 1hr 30min ('''No shaking'''). '''Prepare W-buffer in a 1.5-mL clear low-binding tube''' 1rxn ----------------------------- H2O 395 0.1% SDS 50 2M NaCl 45 2M Tris 5 0.5M EDTA 5 ----------------------------- 500 *Adjust to incubator to 48C and pre-warm the washing buffer. (prewarmed at 48C w/ some little mixing) *Pellet the labeled bacteria (10,000xg, 5min at RT). Remove the supernatant carefully. **Pallet still substantial (barely any change in size since last PBS washing) *Resuspend the pellet with 500 uL washing buffer by repeat pipetting. Incubate the mixture at 48C for 20min without shaking. **Had less than expected amount of W-buffer so added 500ul W-buffer into one tube then <500ul into the other tube *Pellet the labeled bacteria (10,000xg, 5min at RT). Remove the supernatant. *Additional washing with 500uL 1X PBS twice '''(10,000xg, 8min)''' **After last wash, still substantial pallet so can still see it clearly in tube *Resuspend the pellet with 100uL PBS and stored in 4C fridge ==Results== ===Microscope Images=== **ETOH MB PU (124/248) [[File:10_17_2010_3_Chris.jpg|500px]] **RLV MB PU (124/248) [[File:10_17_2010_5_Chris.jpg|500px]] Results based on microscope images show that RLV-fixed mouse bacteria showed slightly better fluorescence than ETOH-fixed ===Flow Cytometry Results=== *ETOH E. coli PN (used to calibrate flow cytometer PMT for ETOH E. coli) [[File:002.jpg|500px]] *ETOH E. coli PU [[File:004.jpg|500px]] *ETOH MB PN (used to calibrate flow cytometer PMT for ETOH MB) [[File:003.jpg|500px]] *ETOH MB PU [[File:006.jpg|500px]] *RLV MB PN (used to calibrate flow cytometer PMT for RLV MB) [[File:10_17_2010_001.jpg|500px]] *RLV MB PU [[File:005.jpg|500px]] ===Overall Conclusions=== *E. coli PU doesn't seem like it's working, but still move on because successful cultured mouse gut bacteria hybridization *Based on both microscope and flow cytometry results, RLV-fixed MB has higher signal than ETOH-fixed MB
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