The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that’s another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall off when my first attempts failed (and by first, I mean a month of failure). Because I was a beginner in this technique, my colleagues gave useful suggestions and advice about what could have gone wrong. Below is a simple checklist to use when you don’t see those tiny and precious colonies:
Did You Properly Follow the Transformation Protocol?
First and foremost:
If you are a beginner (just like me), the protocol is the first thing to double check. Usually, laboratories have their own protocols that are passed to you once you start working there. But, are those accurate and up to date? I know, you are too scared to ask your P.I. or even the person who passed you the protocols. For the sake of your experiment, though, always do a quick literature/protocol review to see whether the protocol you have is consistent and correct.
Did You Adhere to the Protocol?
Once you checked this, examine each step: Did you follow the timings properly? Sometimes you don’t have to strictly adhere to the indicated time. For example, the time your bacteria spend at 4°C, you can keep them there for longer than the protocol says. This applies to the final incubation at 37°C, as well, because your bacteria have already taken up the plasmid (in the best case scenario) and are initiating the antibiotic resistance protein expression. However, you must strictly follow the heat shock timing. The incubation time depends on the competent cells and protocol you are using, but it’s usually 30 to 90 seconds at 42°C in a water bath.
Are Your Competent Cells Alive and Viable?
Once you’ve checked that you followed all of this properly, let’s analyze the next possible explanations:
The most important thing to think about if you see no colonies after transformation are your competent cells. Whether you prepared them in the lab or bought them, always check (especially if you prepare them) that they are alive. Do this quickly by inoculating an LB plate with your competent bacteria. If you see colonies after overnight incubation at 37°C, then you are good to go! While checking viability, also transform your cells with an uncut plasmid and calculate the transformation efficiency. You should expect a transformation efficiency higher than 104.
Did You Add the Correct Amount of Plasmid DNA?
Okay, is everything going alright up to now? Let’s analyze the next scaffold:
Sometimes, because we are following THE protocol, we forget to check our plasmid DNA concentration. If you find yourself thinking “I just add 5 µL of the ligation mixture” and have no clue what went wrong, you might begin to suspect your plasmid DNA concentration. Trust me, it’s not about the volume of DNA you add to the reaction mix, but the amount of it. 20 to 100 ng of plasmid DNA in total works just fine!
Are Your Plates Supplemented with the Correct Antibiotic?
Can you guess what’s next? Your resistance selection, exactly!
Antibiotic resistance is the most commonly used selection marker in bacterial transformation and that’s the one I’m going to focus here. However, this applies to any selection marker you use. Adding the wrong antibiotic (i.e., you add kanamycin instead of ampicillin) is not unusual, and this tends to happen when you are in a hurry or the antibiotic was misplaced and you ended up taking the wrong one. Keep an eye on this point! Finally, do not forget to verify the antibiotic concentration in your plates. If you are still not sure about this one, just inoculate one of your plates with your competent cells. If they do not grow, then your plates are perfect—with the correct antibiotic, of course!
This is a simple but effective start to troubleshoot your transformations. If you have gone through this entire checklist and still do not see those colonies in your plates: Do Not Panic! Sometimes your bacteria are testing you—and I’m not joking. Do it again!Image credit: MadLab Manchester Digital Laboratory