Contribution | iGEM Concordia-Montreal

Contribution

Troubleshooting

Colony PCR is an important technique used for detecting whether or not a gene has been inserted successfully. We spent a lot of time perfecting our colony PCR protocol due to many failed experiments when building our reporters. Therefore, we decided to develop a troubleshooting guide based on the different parameters we adjusted. The colony PCR troubleshooting guide is organized into three sections: sample preparation, thermocycler settings, and reagents.

All potential troubleshooting solutions were organized in order of priority. Sample preparation requires the least amount of adjusting, whereas changing the reagents requires purchasing new components, and therefore should be a last resort.

1. Sample Preparation

1.1 Cell Density

A non-optimal cell density of the sample can cause a failed PCR reaction.

Table 1: Sample Preparation
	Detail
Cause	Not enough cells -> Causes many primer dimers since there are more primers than DNA. Too many cells -> More likely the DNA will reanneal (and not amplify) since there is more DNA than primers.
Solution	Adjust the density of cells in the samples.

Figure 1: Sample Preparation Solution

Tube 1: Water
Tube 2: Not enough cells
Tube 3: Right amount of cells
Tube 4: Too many cells

2. Thermocycler Settings

2.1 Extension Time

The primer extension time depends on the length of the DNA sequence being amplified.

Table 2: Extension Time
	Detail
Cause	If the DNA sequence is long, a lower primer extension time may not allow for the entire sequence to be transcribed.
Solution	Increase the extension time.

2.2 Annealing Temperature

Each primer has its own unique annealing temperature.

Table 3: Annealing Temperature
	Detail
Cause	The annealing temperature being used may not be appropriate for the primers being used.
Solution	Do a temperature gradient to test which temperature works best with the primers.

2.3 Number of Cycles

The total number of cycles will determine the total quantity of DNA.

Table 4: Number of Cycles
	Detail
Cause	Not having enough cycles could result in not enough DNA being amplified to be detectable on an agarose gel.
Solution	Increase the number of cycles.

3. Reagents

3.1 Primers

Primers are specific to the region being amplified.

Table 4: Primers
	Detail
Cause	The primers themselves may not be specific enough to the region of interest. If the primers are too short, they may not be specific enough, which can lead to off target binding..
Solution	Use primers that are longer.

3.2 Polymarase

There are different polymerases that can be used for PCR.

Table 5: Polymerase
	Detail
Cause	Different polymerases have different efficiencies, so some may work better for some reactions and not others.
Solution	Try using a different type of polymerase.

Science Communication Contribution

While full guides provide extensive details and are helpful, our team believes that one-page cheat sheets highlighting the key points of a particular subject can be beneficial and less daunting to go through.

Figure 2: SciComm Cheatsheets

Your Research, 3-Min Cheat Sheet

A 3-Minute Thesis can be challenging for anyone, especially when your research has taken hours of work and the academic paper is numerous pages long. To be able to condense everything into 3 minutes and get your point across to your audience takes some practice, which is why we created a summary sheet with some tips that students can quickly consult.

Scientific Presentations Cheat Sheet

Presenting plays a major role when defending a thesis or attending conferences. However, many undergraduate students do not have the opportunity to develop these skills in advance. For this reason, we decided to make a fun cheat sheet for students to use in order to better organize their presentation content and some tips to consider when presenting.

Team:Concordia-Montreal/Contribution