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='''Non-fixed E.coli cell cryopreservation test using Glycerol or Betaine'''= ==Objective== *In my test on 04-26-'11, I found that using fresh cell is significantly helpful to SYBR-GOLD or DAPI staining. YOYO-staining is not working probably due to the staining method is not optimized well. *In order to prepare samples for long term usage, I am trying to cryopreserve them in -80C using Bigelow lab protocol. *Cyropreservation protocol from Bigelow's lab: #[[Media:Bigelow_lab_Sample_cryopreservation_glycerol.pdf|Sample_cryopreservation_glycerol]] #[[Media:Bigelow_lab_Sample_cryopreservation_BeTE.pdf|Sample_cryopreservation_BeTE]] ==10% Glycerol cryopreservation== #Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes #Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL 1X PBS(0.2um-filtered). #Transfer 100uL suspension into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. #Leave the cell pellet on ice. #Prepare 50% glycerol in H2O(adding 20mL H2O with 20mL 100% glycerol in a 40mL tube). #Resuspend cell pellet with 500uL 1X PBS(0.2um-filtered) #Add 125uL 50% glycerol into the 500uL cell suspension. Repeat pipetting slowly 10X using 1000p tips. ##The final concentration is 125x50% / (125+500)=10% #Store the cell in -80C. Labeled as '''E.coli + GC'''. ==0.5M Betaine cryopreservation== *Prepare following working solution #8X TE buffer: Add 4mL 50X TE with 21mL H2O. #Betaine working sol (4M).: Dissolve 4.8g Betaine (MW: 117.15) with 8mL 8X TE. Filtered the Betaine solution through 0.2um filter. Add more 8X TE to final volume at 10mL. ##It is supposed to add 8X TE buffer to a final volume of 10mL. However 4.8g Betaine is very bulky so the volume exceeded 10mL even I just added 8mL 8X TE buffer. ##The final concentration is 4M (4.8/117.15/0.01). #(Using the same batch of cell pellet from the test above)Resuspend cell pellet with 500uL 1X PBS(0.2um-filtered). #Add 71.5uL filtered Betaine solution into the 500uL cell suspension. Repeat pipetting slowly 10X using 1000p tips. ##The final concentration is 0.5M (4x71.5/(500+71.5)) #Store the cell in -80C. Labeled as '''E.coli + BT'''. ==Validation== *I will compare the cryopreserved cells with fresh cells using my standard DAPI staining protocol. [[File:050311-Pos-BF-10X.png|400px]] [[File:050311-Pos-DAPI-10X.png|400px]] [[File:050311-GC-BF-10X.png|400px]] [[File:050311-GC-DAPI-10X.png|400px]] [[File:050311-BT-BF-10X.png|400px]] [[File:050311-BT-DAPI-10X.png|400px]] *Although it looks brighter on BT-treated E.coli cells, it is due to the auto-contrast setting. *I also captured images using fixed contrast (low:100, high:300). The GC treated E.coli and BT treated E.coli showed very similar fluorescent intensity. *Either Betaine or Glycerol works fine to cryopreserve the fresh E.coli for long term usage of DAPI staining. ==Test of loading cell on PDMS microwell (provided by Jeff)== *Newly fabricated PDMS microwell (id: 400um) appears to be an ideal sample reservoir for the micro-manipulation. *The smaller volume should help to restrain the floating of E.coli cell in suspension, which is beneficial to target capturing. *Possible isolation approach: #Dilute the E.coli cells into desired density. #Transfer E.coli cells into microwell. #Identify the wells containing only single E.coli cell. #2nd transferring from microwell to PCR tubes. ===Procedures=== *Since the PDMS microwell is very thick, it should be put on another station for microscopy. *Using 4X objectives for observation. ===Results=== [[File:Jeff-PDMS-microwell-BF-4X-new.png|400px]] [[File:Jeff-PDMS-microwell-DAPI-4X-new.png|400px]] [[File:Jeff-PDMS-microwell-DAPI-4X-washed2-new2.png|400px]] ===Discussion=== *Compared to SU-8 based microwells, PDMS-based microwells has less autofluorescence problem and can serve as good sample reservoirs. *After washing microwell using 100% isopropanol, I remove most of dusts but the well are still not clean enough for microisolation. ==Next== *The non-fixed E.coli cells are ready for real isolation tests. New samples will be filtered with 5um, mixed with glycerol, and then isolated under a clean working condition.
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