Dear All,
What is the best way of picking the positive clones while establishing a monoclonal cell line expressing your desired transgene. I am currently doing it with a P-20 pipette under a light microscope. However, it is very hard to tell whether I have specifically picked the single clone or not. Is there a better way.
Thank you
Ikram
Hi,
I suggest using "cloning rings" and silicone. Just plate your positive transfected cells on a 15 cm dish (if growing in adhesion, I assume) at very low density. Follow growth of cells. When a clone start developing a "colony" (about 100 cells), under the microscope mark the bottom of the petri dish with a marker pen. Then on the hood, apply silicone to the base of a cloning ring and isolate the colony from the rest of growing cells. Remove medium, wash with pbs and using trypsin detach cells as usual. Transfer the colony in a 96 well plate and continue growing other clones. Keep taking cells as some clones will certainly die during picking up. You can leave or remove the ring in the petri dish.
If your cell line grows in suspension, dilute your transfected cells as much as you can (ideally 1 for each well of a 96-well plate...), but you will not get a monoclonal population as cells growing in adherence. Very difficult. You will get a polyclonal population that should "abundantly" express your transgene if compared with control.
Hope that may help. Good luck
Regards,
Daniele
Hello,
the best way is by using a several standard dilution. Start by doing a solution at 1.106cells/mL and then dilute it until reach 1cell/100uL (like 10 000cells/ml then 100cells/ml then 1cell100ul). Take care to well homogeneize before every pipeting. Then seed you cell in a full 96-wells plate at 1cell per well (by adding 100ul of your last dilution). The next day take time to watch every well (some will have 2 cells some will be empty). When the selected 1cell well reach confluency, expand cell by increasing the area of culture (p48, p6, t25 and then t75).
good luck
Dear Dr Baiz and Dr Ferrandon
Thank you very much for your suggestions. I appreciate your help.
Thank you
Ikram
Sylvain Ferrandon - I have a question and do not want to start a new topic.. You suggest to expand from 96 -48-6-T25-T75.... Is this particularly important for minimizing number of cell divisions a cell goes through? Or is there any other reason? I am currently doing this but I have been asked why I am not going directly from 48 well plate to T25/T75. As this will reduce the passage numbers of the cell line and I can freeze vial of cells earlier. Thanks.
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