Some Thoughts On Creating Stable Cell-Lines

Creating stable cell-lines can be a straightforward or tricky process. In this post I will share some techniques or quirks, which I found worked when creating stable cell-lines.

* Use cells with low passage number – Thaw out your cell stocks^1,2 and start with “new” cells with low passage number.

* Culture cells in antibiotic-free media – By cutting out penicillin/streptomycin, I found increases in my transfection efficiencies. It also avoids any possibility of the penicillin/streptomycin interfering with the selection antibiotic during the selection process.

* Make the selection media fresh – Have aliquots of your selection antibiotic at a higher concentration and dilute it into fresh media each time you need to do a media change.

* Change the selection media daily – After transfection, change the selection media daily up until you reach the limiting dilution stage. It may seem excessive or wasteful but I have found that by having freshly made selection media and a daily media change ensures that the untransfected cells are effectively killed off.

* Have high concentrations of your antibiotics aliquoted – Freeze-thaws and heat-cool cycles can affect the efficacy of some antibiotics so to avoid any issues calculate the approximate amount that you will need for selection and aliquot small amounts into separate tubes.

Cloning Tip – How Not To Expose Inserts To UV During Preparation

As mentioned in a previous post¹ there is a way that you can isolate PCR-amplified inserts from DNA agarose gels without exposure of the insert to UV.

First, create replicates of your insert by amplifying multiple PCR reactions.  I generally prepare between 3-5 replicate reactions to increase my insert yield. Prepare a 1% agarose gel + ethidium bromide. After your PCR goes to completion, cool the reactions to 4 degrees or leave it on ice. Add loading dye and load the reactions onto your gel. In the lanes indicated with the letter “L”, add your DNA ladder. I have used a 100bp ladder as an example.

Run the gel. Once completed, remove the gel from the tank and cut the gel as indicated by the red dashed line. Take the smaller portion of the gel to a dark room and visualize on an open UV box. Take a blade or scalpel and mark the gel by nicking the gel edge just above and below the band of interest (green) as indicated by the asterisks. 

Reassemble the entire gel at your bench and then using the nicks made to the gel and the ladders as a guide, carefully cut out the area where your replicate bands of interest are likely to lie as indicated by the dashed red lines.

Gel purify the gel cut-outs containing your bands of interest (green) and proceed with your cloning protocol.

Cell Culture and Antibiotics

There are arguments for and against the use of antibiotics (e.g. penicillin/streptomycin) in cell culture. On the one hand, it is a useful prophylactic to prevent cultures from contamination by providing a layer of protection. However, arguments have been made against its use with claims that constant usage allows for unintentional selection of antibiotic resistant bacteria, researchers become reliant on it and becoming lax in applying aseptic techniques and good cell culture practice.

I am inclined towards the latter view of avoiding the use of antibiotics, but mainly because antibiotics can interfere with particular experiments, namely transfections. However, I do find that it is better to use aseptic techniques because it is better to know quickly that your cells are contaminated rather than have low level contamination hang around which is not visible. The danger of low level contamination is that it will affect and alter your cells and interfere with your experiments.

In regards to students or researchers learning cell culture for the first time, it is probably better for them to see how lapse in aseptic practice can easily lead to contamination. People learn from their mistakes and will only improve with practice.