For scientists, the ability to troubleshoot their experiments is a valuable skill to develop. This skill allows you to be an independent and responsible researcher (Roberts, 2001).
Although important, troubleshooting as a subject is commonly not included in many undergraduate molecular biology courses. Therefore, this article provides some useful steps to troubleshoot problems in the molecular biology laboratory.
Some common steps for troubleshooting problems in the lab are (Gerstein, 2004):
1. Identify the problem
2. List all possible explanations
3. Collect the data
4. Eliminate some possible explanations
5. Check with experimentation
6. Identify the cause
Below, we present two different scenarios and how this troubleshooting process could be applied. While these scenarios are very specific, this troubleshooting approach can be used broadly across other experiments in your lab.
Example 1: No PCR Product Detected
1.Identify the Problem
First, you need to identify the problem, but try not to define the cause yet. In this example, let’s say you don’t see any PCR product on the agarose gel. You see the presence of your DNA ladder, so there is no problem with the gel electrophoresis system.
Now, you identify that the problem is your PCR reaction. Remember, we’re not looking at the cause just yet.
2.List All Possible Explanations
After you identify the problem, list all the possible causes for the issue (in this example, your PCR reaction). Start listing the obvious causes. These could be each of the ingredients in your PCR Master Mix: Taq DNA Polymerase, MgCl2, Buffer, dNTPs, primers, and your DNA template. After listing the obvious causes, include the causes that might have escaped your attention, such as the equipment and the PCR procedure.
3.Collect the Data
To do this step, start collecting data for the easiest explanations. First, check if the PCR equipment works properly. You can ask other scientists in your laboratory if they have encountered similar problems. However, if there is no problem with the equipment, then go ahead and collect data for the other explanations.
If you ran your samples with all proper control reactions, find out whether your positive control (using a DNA vector as the DNA template) was present in your gel or not.
Storage and Conditions
The next step is to find out about the expiration date of the PCR kit that you used. In addition, check if your PCR kit was stored according to your vendor’s instructions.
To collect data about your procedure, check your laboratory notebook for the procedure that you used in the experiment and compare it with the manufacturer’s instructions. Write down all the modifications you made during this experiment, and note any possible missed steps.
Based on the data you collected, eliminate the explanations that you have determined are not the cause. For example, if your positive controls worked, your kit had not expired and it was properly stored, you can eliminate your kit as the possible cause. If you also find out that you didn’t modify the PCR procedure, eliminate it as the possible cause.
5.Check with Experimentation
Recheck your list and design an experiment to test the remaining explanations. For example, test whether your DNA templates are the possible cause. For example, run the DNA samples on a gel to see if there is any degradation. Measure the DNA concentration to see if you used enough template for your PCR reaction.
6.Identify the Cause
The last step is to eliminate most of your explanations that you’ve ruled out and identify the only one remaining as the cause. Using information from the experiment you just ran (in step 5), plan ways in which you’ll fix the problem and redo your experiment.
If this issue is something that could arise again, you might want to find ways to reduce future errors. For example, rather than making your own master mix, you could use a premade master mix.
Example 2: No Clones Growing on the Agar Plate
1.Identify the Problem
First, check all the transformation plates and see if any colonies are growing on your control plates. If there were colonies on your plates, then the problem is the transformation of your plasmid DNA.
2.List all possible explanations
After you identified that the problem is not the competent cells, the possible explanations for your failed cloning may be your plasmid, the antibiotic, or the temperature during heat shock procedure.
3.Collect the data
If you included your controls in your transformation, your positive control plate should be the cells transformed with an uncut control plasmid. This plate should contain many colonies. If there are only few colonies growing on this plate, the efficiency of the competent cells may be too low.
To find out if your antibiotic is the cause, check if you used the correct antibiotic for selection and the concentration recommended for selection.
To see if the incorrect temperature during the heat shock could be the cause, find out if the temperature water bath was at 42˚C.
Now you can start eliminating some of the possible explanations. For example, based on your data collection, there were many colonies growing on your positive control plate. It means that your competent cells were efficient.
You also found out that you used the correct antibiotic with the recommended concentration for selection. Then, you can eliminate antibiotic as a possible cause.
Moving on to the temperature during the heat shock, you found out that the temperature of the water bath was at 42˚C. Therefore, the procedure was not the problem and this should be eliminated from your list.
Now the only possible cause is your plasmid DNA.
5. Check with Experimentation
In order to test if your plasmid is the problem, check if it is intact using gel electrophoresis and the plasmid concentration is not too low. In addition, check your ligation by sequencing your plasmid to make sure the insert DNA is in the plasmid. Make sure you follow the instruction from your transformation protocol regarding the concentration of plasmid you should use.
6.Identify the cause
For the last step, gather all the information you need after you ran the experiments. For example, you made sure that there was no problem with your ligation based on your sequencing data, but you saw a faint band on the gel electrophoresis and found out that the concentration of your plasmid was too low. Therefore, you identified that the cause of your failed transformation was that your plasmid DNA concentration was too low.
To better organize your troubleshooting process, below is a checklist you can use:
Gerstein, A. S. (2004). Molecular biology problem solver: a laboratory guide: John Wiley & Sons.
Oelker, S. (2012). LibGuides: Biological Sciences: FAQs.
Roberts, L. M. (2001). Developing experimental design and troubleshooting skills in an advanced biochemistry lab. Biochemistry and Molecular Biology Education, 29(1), 10-15.