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.
|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.
|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.
|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.
|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.|
Primers are specific to the region being amplified.
|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.|
There are different polymerases that can be used for PCR.
|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
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