Sam:LabNotes/Micro-manipulation/2011-4-29: Difference between revisions

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>Sam Chiang
>Sam Chiang
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  [[File:BT-BF-10X.png|400px]]  [[File:BT-DAPI-10X.png|400px]]
  [[File:BT-BF-10X.png|400px]]  [[File: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

Revision as of 21:21, 5 May 2011

Non-fixed E.coli cell cryopreservation test using Glycero and Betaine

Objective

  • In my test on 04-26-'11 test, 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

Glycerol cryopreservation

  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.
  8. Store the cell in -80C. Labeled as E.coli + GC.

Betaine cryopreservation

  • Prepare following working solution
  1. 8X TE buffer: Add 4mL 50X TE with 21mL H2O.
  2. Betaine working sol.: Dissolve 4.8g Betaine with 8mL 8X TE. Filtered the Betaine solution through 0.2um filter.
  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.
  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:Pos-BF-10X.png  File:Pos-DAPI-10X.png

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