help - stable transfection clone pick up - (Apr/08/2004 )
To pick up clones, I have followed the cheapest and I think efficient way. What you have to do is to take pasteur pipettes, which we generally use in tissue culture to suck the media, and break it at the bottom (so you will have 3-4cm length hollow glass cylinder).
Autoclave those cylinders. Mark your colonies at the bottom of plate by viewing in microscope and then just put the cylinder on one clone. Put a drop of trypsin and after some time serum containing media. Use P-200 to break the clumps and transfer the clone to 98 well plates. I followed this method to establish the stable cell lines and found aseptic and efficient. Best Luck.
Hello All
After 4 weeks of selection I want to transfer my clones from the dish to well plates, but I wouldn't liko to trypsinized them, nor I have the clone cylinder. I have heard that I could do the transfer putting a kind of paper on the selected cells. Has anoyne experience on that?
Thanks
The paper method is a piece of filter paper with trypsin on it. You set it on the colonies and then after a set amount of time, lift it up, hopefully with cells sticking to it, then rinse it in media and plate or some such thing.
If you are set on avoiding trypsin, you can always just scrape up the colony with a micropipettor. You will damage many of the cells, but it is a clone, so theoretically if some survive you are money.
Personally, I don't think the trypsin is going to be disadvantagous.
Beverly
If you are set on avoiding trypsin, you can always just scrape up the colony with a micropipettor. You will damage many of the cells, but it is a clone, so theoretically if some survive you are money.
Personally, I don't think the trypsin is going to be disadvantagous.
Beverly
Thanks Beverly,
you were very helpful
When I clone, I plate my cells in a dish with a removable lid and when you have some colonies, write on the bottom of the dish with a sharpie while looking at the colony under the microscope to make sure you are marking just one. (Takes a bit of practice to line up the sharpie under the scope) I rinse the dish with a little bit of trypsin and then add some new trypsin, just enough to make the bottom of the dish wet. Then I use a 1mL pipetteman and suck up the colony marked by the sharpie, much easier than looking for colonies when you are working in the hood. Just look for the colored dots. I have had real good luck with this in the past and it cuts out all that putzy crap with the rings and the discs and whatnot.
Beverly
Hi Beverly,
I am making stable cell lines for the first time and therefore was wondering if you keep them in the selective media after the 1.5 weeks or if you give them a break at some point to recover from it?? The cell line we are using is new and the the cells are very sensitive, so even if I use 50ug/ml G418 they are very unhappy, therefore I would like to switch to normal growth media since all my control cells are already dead. Do you think that 1.5 weeks are not sufficient to get stable transfections??
Thanks,
Julia
jjr,
When I make stable expressing cell lines, the first thing I do is a toxicity curve on the cell line. Treat with increasing concentrations of G418 until all the cells are dead. The lowest concentration that kills all the cells in a week or so is the concentration you want to use.
Then when you do your transfection always include a dish that gets only the transfection reagent and no DNA (a mock transfected control). Then when you treat your cells with G418 to isolate the cells which have taken up the plasmid, you know that the cells you have still alive in the plasmid dishes contain the resistance marker after your mock transfected dish is dead. (A little note, there is always one colony left in the mock transfected control. I suppose some sort of mutations can occer or something. No matter what, you can always look hard and find some cells that are still alive. Vexing, but doesn't seem to interfere with the outcome of the experiment.)
At this point, I usually keep the G418 on for a couple of passages, as I think some of the cells might be protected by their neighbors and breaking them up allows the G418 to get to all the cells. Then you can be certain your plasmid is incorporated into the genomic DNA. (After several passages any cytoplasmic plasmid will be essentially lost.) Once the plasmid is in the genomic DNA, there is no way for the cell to get rid of it. Sometimes I sequence the genomic DNA, just to make sure it is there. Just make primers that bridge the gap between your plasmid sequence and your insert sequence. If you get bands, the plasmid is there.
One other thing, if you linearize your plasmid prior to transfection, you know where the cut is going to be and you can avoid having it in the coding sequence. It you stably transfect with a circular plasmid, you don't know where the plasmid will be cut to go into the genomic DNA.
Hope this is all coherent. It's Monday. If you have more questions, just reply.
Beverly
Hi,
I think that 50 ug/ml of G418 is too low, you probably do not have transfected cells. The best method for stable transfection is to electroporate linearized vector and to start selection already 24h posttransfection. The efficiency is critical, I have good experience with GTporator solution. After two weeks pick up clones by pipet (10 ul), they should be visible by naked eye. Depending on the posible overexpressed protein toxicity, I would recommend to keep selection media all the time.
L. Burysek
Did anyone know where to get silicone gel?
I bought some silicone gel from Walgreen, then after autoclaving, all of them were siliconized...
Thanks..
KY
Does anybody has the experience in stable transfection with lipo-based transfection reagents in the mES cells? I'm a beginner in this work.I have questions as following:
1)if I do a G418 killing curve, what range of the concentrition woud be best?
2)timecouse, how long should a lowest concentrition of G418 kill all the untransfected cells according to your experience?
Thank you!
I think that 50 ug/ml of G418 is too low, you probably do not have transfected cells. The best method for stable transfection is to electroporate linearized vector and to start selection already 24h posttransfection. The efficiency is critical, I have good experience with GTporator solution. After two weeks pick up clones by pipet (10 ul), they should be visible by naked eye. Depending on the posible overexpressed protein toxicity, I would recommend to keep selection media all the time.
L. Burysek
If you are set on avoiding trypsin, you can always just scrape up the colony with a micropipettor. You will damage many of the cells, but it is a clone, so theoretically if some survive you are money.
Personally, I don't think the trypsin is going to be disadvantagous.
Beverly
I have tried this method but the cells didn't grow up.
How can I improve the efficiency of picking up the colonies?
Thanks!
I found very easy to wash the plate with PBS (or versene), take 20ul of trypsin and trypsinize directly the clone. After that I've put it in a well of a 24 well and then I scaled up the dishes to amplify.