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        Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates

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        Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates the Laboratory of Dr. Allan Bradley
        Baylor College of Medicine, Houston, Texas 冻存96空板中的胚胎干细胞克隆 

        Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates
        
        
         1. Refeed cells 2-3 hours before use.
        
         2. Check each well for confluence and number of clones.  
                    Selectively passage clones which have less than 10 
                    colonies per well to another multiwell plate.
        
         3. Aspirate off all of the media and wash 2 times with PBS.
        
         4. Using the multi-channel pipetter, add 50 ul of trypsin 
                    to each of the wells.  Change pipette tips between wells!
        
         5. Incubate @ 37o C for 10-15 minutes.
        
         6. Add 50 ul of 2X Freezing Media (60% DMEM, 20% FCS, 20% DMSO) 
                    to each well and break up the colonies by pipetting up-and-down 
                    about 5 times.
        
         7. If required, remove 25 ul of the cell suspension and 
                    pipette directly into a new multiwell plate.  If the 
                    cells are being grown up for Southern analysis, use a 
                    pre-gelatinized plate containing 200 ul of fresh M15 media 
                    per well.  Alternatively, if the clones are being screened 
                    using PCR, use a 96-well plate containing PCR Lysis Buffer 
                   (50 ul + Proteinase K), lysing the cells either individually 
                   or in appropriately mixed pools.
        
         8. Using the multi-channel pipetter, add 100 ul of 
                    filter-sterilized (0.22 um) Light Paraffin Oil to each well.  
                    This prevents degassing and evaporation during storage @ -70o C.
        
         9. Replace the lid on the plate and secure by completely sealing 
                    with tape around all the edges.  Place the plate in a 
                    polystyrene cuvette box, cover with a polystyrene lid 
                    that fits, secure with tape, and freeze @ -70o C (the 
                    temperature optimally should drop approximately 1o C/minute).
        
         10. For samples being screened by PCR, take the plate of 
                    cells in PCR Lysis Buffer (prepared in step 7, above) and 
                    place @ 55o C for 1 hour to overnight.  Adequate steps must 
                    be taken to avoid evaporation (e.g., addition of oil to 
                    cover each well and/or incubation in a humidified chamber, 
                    such as a sealed plastic container with a wet sponge inside).  
                    After incubation, the lysates may be stored @ -20o C.  
                    However, prior to use for PCR, the Proteinase K must be 
                    inactivated by transferring the lysates to labelled 0.5 ml 
                    Eppendorf tubes and heating @ 94o C for 15 minutes 
                   (Program #99 in the PCR machine).
        
         11. To retrieve ES cell clones that have been frozen by this  
                    method, take the 96-well plate from the -70o C freezer and 
                    place directly into the 37o C incubator.  Allow all of the 
                    wells to thaw completely (this may take 10-15 minutes for 
                    the wells near the center of the plate), then remove the 
                    clones from the wells and transfer to appropriately labelled 
                    wells in 24-well feeder plates pre-equilibrated with 2 ml of 
                    M15 media per well.  For maximum recovery of sample, it is 
                    important to vigorously pipette the thawed cells to dislodge 
                    them from the bottom of the plate (where they settle during 
                    the freezing process).  Since the cells tend to accumulate 
                    around the perimeter of the wells as a consequence of the 
                    minimal freezing volume, rinse the wells with media to further 
                    facilitate maximum recovery and transfer to the appropriate 
                    wells in the 24-well feeder plates.  There is no need to remove 
                    the DMSO and the paraffin oil until the cells have replated 
                   (24 hours after passage).
        
        
        From the Laboratory  of Dr. Allan Bradley               
        Baylor College of Medicine, Houston, Texas
        
        

         

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