Bacterial transformation can also be performed using electroporation, but this method is less common. It runs on the same principle as heat shock protocol, but electricity is used instead of heating the bacteria. Electroporation sometimes results in more transformed colonies, but not always. Once transformation has occurred, it’s time to pick colonies after transformation. Colony picking protocol isn’t too difficult but is quite an important process of bacterial transformation. Read on to learn about the precise protocol when picking colonies.
Description of Picking Colonies After Transformation
Once transformation is complete, you will be picking colonies from agar plates.
You should have three bacterial plates to choose from:
- The ligation plate.
- A plate with no fragment but with ligase (control plate).
- A plate with no fragment or ligase.
The ligation plate will have myriad colonies, and the other plates will have less. It’s important to note that when you pick colonies after transformation, it is chosen by the ratio of colonies you have on your control plate (the one with ligase) to the number of colonies on your ligation plate.
Here is an example if you are performing standard sticky-ended directional cloning. If you have ten times more colonies on the ligation plate compared to the control plate, 9 out of 10 colonies should likely have the correct fragment ligated into it.
However, there are bumps in the road sometimes when picking colonies after transformation, such as multimers or concatemers. Because of this, you’d likely pick five to ten colonies, depending on outside factors.
In contrast, if you only have two times more colonies on the ligation plate than the control plate, you would pick ten colonies or more. You can always place plates back in the refrigerator and redo the process. Unless your ligation is very complex, you wouldn’t pick a large number of colonies, such as 50. It will just slow you down.
When picking colonies after transformation, an enzyme fails to work in some cases. This can result in no difference between the ligation and control plates. If this happens, you’ll have to start the entire process over. Some tips to prevent this from happening include:
- Decreasing the background
- Digesting for longer
- Exposing to less UV
- Using someone else’s enzymes
- Dephosphorylating for longer
There is a certain protocol when it comes to the act of picking colonies after transformation using an automated colony picker or performing the process manually.
What Is the Protocol for Picking Colonies After Transformation?
The protocol for picking colonies after transformation must be performed in a sterile environment, typically with a Bunsen burner or class 1 hood nearby. It is very important not to cross-contaminate. The protocol is:
- Take the plates out of the incubator (which should be 37°C), upside down. This is the same way you have put them in. Place them on the benchtop.
- Have a pipettor handy. You must pipette 3 to 5 mL of LB media (catalog numbers L3522, L2542, or L3022), with the correct concentration of antibiotic into 25 to 60 mL tubes. The amount of air is also important. There should never be more than a 1:3 ratio of liquid to air. Ideally, the ratio should be much higher.
- Next, place a sterile pipette tip on another pipette with one hand. Using your other hand, pick up the upside-down ligation plate.
- Turn the plate over, and touch the sterile pipette tip to a bacterial colony that is away from other growth.
- Place the pipette tip with the bacteria on it into one of the LB tubes. Gently move the tip so that the bacteria is released into the liquid.
- Once all of these steps are complete, you must culture the tubes overnight in an incubated orbital shaker, which should be 37°C and 190 to 225 rpm.
You can also freeze colonies during the process of picking colonies after transformation. Colonies in liquid can be viable at 4°C for several weeks.
Other things to consider:
How can we optimize the colony picking protocol after transformation to ensure efficient selection of colonies with the correct fragments while minimizing the risk of selecting multimers or concatemers?
Maximizing the efficiency of our colony picking protocol post-transformation is paramount. We need to ensure that we’re selecting colonies with the correct fragments while minimizing the risk of encountering multimers or concatemers. By fine-tuning our picking strategy and perhaps adjusting the ratio of colonies selected based on the control plate comparison, we can optimize the protocol for more accurate results and streamlined processes.
What strategies can we implement to mitigate the risk of enzyme failure during colony picking after transformation, particularly when there is no discernible difference between the ligation and control plates, to avoid restarting the entire process?
Addressing the possibility of enzyme failure during colony picking after transformation is crucial to avoid setbacks and delays. Implementing proactive strategies to mitigate enzyme failure, such as adjusting background levels, optimizing digestion times, and exploring alternative enzyme sources, can help prevent instances where no difference is observed between ligation and control plates. By taking these precautions, we can minimize the need to restart the entire process, saving time and resources.
How can we effectively integrate automated colony picking solutions into our laboratory workflows to enhance efficiency and accuracy in the process of picking colonies after transformation? What are the key considerations and potential benefits of adopting automated systems in this critical aspect of our workflow?
Incorporating automated colony picking solutions into our laboratory workflows presents an opportunity to enhance efficiency and accuracy in the colony picking process post-transformation. By seamlessly integrating automation technology, we can streamline the picking process, reduce manual errors, and increase throughput. Exploring the benefits and considerations of adopting automated systems will enable us to make informed decisions about integrating this advanced technology into our workflow to optimize our laboratory operations.
Hudson Robotics has been the leading supplier of automated products for over 38 years. To learn more about picking colonies after transformation, speak to a representative today!