Sam:LabNotes/Micro-manipulation/2011-4-29

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Non-fixed E.coli cell cryopreservation test using Glycerol or Betaine[edit]

Objective[edit]

  • 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:
  1. Sample_cryopreservation_glycerol
  2. Sample_cryopreservation_BeTE

10% Glycerol cryopreservation[edit]

  1. Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
  2. Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL 1X PBS(0.2um-filtered).
  3. Transfer 100uL suspension into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant.
  4. Leave the cell pellet on ice.
  5. Prepare 50% glycerol in H2O(adding 20mL H2O with 20mL 100% glycerol in a 40mL tube).
  6. Resuspend cell pellet with 500uL 1X PBS(0.2um-filtered)
  7. Add 125uL 50% glycerol into the 500uL cell suspension. Repeat pipetting slowly 10X using 1000p tips.
    1. The final concentration is 125x50% / (125+500)=10%
  8. Store the cell in -80C. Labeled as E.coli + GC.

0.5M Betaine cryopreservation[edit]

  • Prepare following working solution
  1. 8X TE buffer: Add 4mL 50X TE with 21mL H2O.
  2. 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.
    1. 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.
    2. The final concentration is 4M (4.8/117.15/0.01).
  3. (Using the same batch of cell pellet from the test above)Resuspend cell pellet with 500uL 1X PBS(0.2um-filtered).
  4. Add 71.5uL filtered Betaine solution into the 500uL cell suspension. Repeat pipetting slowly 10X using 1000p tips.
    1. The final concentration is 0.5M (4x71.5/(500+71.5))
  5. Store the cell in -80C. Labeled as E.coli + BT.

Validation[edit]

  • I will compare the cryopreserved cells with fresh cells using my standard DAPI staining protocol.
File:050311-Pos-BF-10X.png  File:050311-Pos-DAPI-10X.png

File:050311-GC-BF-10X.png  File:050311-GC-DAPI-10X.png
 
File:050311-BT-BF-10X.png  File:050311-BT-DAPI-10X.png

  • 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)[edit]

  • 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:
  1. Dilute the E.coli cells into desired density.
  2. Transfer E.coli cells into microwell.
  3. Identify the wells containing only single E.coli cell.
  4. 2nd transferring from microwell to PCR tubes.

Procedures[edit]

  • Since the PDMS microwell is very thick, it should be put on another station for microscopy.
  • Using 4X objectives for observation.

Results[edit]

File:Jeff-PDMS-microwell-BF-4X-new.png

File:Jeff-PDMS-microwell-DAPI-4X-new.png

File:Jeff-PDMS-microwell-DAPI-4X-washed2-new2.png

Discussion[edit]

  • 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[edit]

  • 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.