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

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>Sam Chiang
>Sam Chiang
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#[[Media:Bigelow_lab_Sample_cryopreservation_BeTE.pdf|Sample_cryopreservation_BeTE]]
#[[Media:Bigelow_lab_Sample_cryopreservation_BeTE.pdf|Sample_cryopreservation_BeTE]]


==Glycerol cryopreservation==
==10% Glycerol cryopreservation==
#Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
#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).
#Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL 1X PBS(0.2um-filtered).
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#Resuspend cell pellet with 500uL 1X PBS(0.2um-filtered)
#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.
#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'''.
#Store the cell in -80C. Labeled as '''E.coli + GC'''.



Revision as of 21:34, 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

10% 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.
    1. The final concentration is 125x50/(125+500)=10%
  8. Store the cell in -80C. Labeled as E.coli + GC.

0.5M 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 (MW: 117.15) with 8mL 8X TE. Filtered the Betaine solution through 0.2um filter.
    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

  • 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