If you are not seeing enough colonies following transformation, a great place to start when troubleshooting is to make sure the cells you are using have high enough efficiency for your experiments. Long or troublesome DNA can lower transformation efficiency, therefore high-efficiency cells would be required to compensate. Intact Genomics competent cells have the highest efficiency cells in the industry (1×10^10 for chemical 5×10^10 electro), so you can be sure to limit these types of issues.
Transformation efficiency is calculated with the following equation:
–(# colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA
To obtain the ‘ng of DNA plated’ you may use the following equation:
–volume of plasmid used in µL x concentration of DNA in ng/µ x (volume plated / total reaction volume)
Rather than calculate yourself, feel free to use our transformation calculator tool in the link below:
Below are a couple of other issues that may cause transformation failure:
- Excessive Freeze thaw – cells should typically be thawed once then used, not reused after thawing.
- Incorrect antibiotic or antibiotic concentration – A common mistake to make, but easily preventable with a diligent experimental setup.
- If you used the correct antibiotic, but still see no or few colonies, check your ligation reaction as you may have inadvertently inserted an incorrect antibiotic backbone.
- Too little DNA transformed – If the DNA you are using is low concentration, you may need to increase the amount of ligation you pipette.
- Heat shock or incubation issues – Heat shock and recovery are vital steps of most transformation processes. If you are using new centrifuge tubes with thicker walls, or defective incubation equipment your experiment may need increased heat shock or incubation/recovery time.
As an alternative, our DirectPlateTM cells eliminate heat shock, lengthy incubations, and time-consuming outgrowth procedures.
Too many colonies, aka a lawn, is another common issue. Below are a couple tips to prevent/remedy this:
- Plate less cells – Intact Genomics cells are so competent, from time to time we recommend plating less volume of cells after recovery or dilute the plasmid that is going into transformation.
- Lack of antibiotic – We are all human, double check you didn’t miss adding the antibiotic to your plates.
- Hot Agar – Be patient when adding antibiotic to Agar, add too soon and the antibiotic will break down.
- Plate making/mixing issues – Make sure to use a stir bar, or if adding antibiotic after the plate has hardened, spread evenly to prevent growth on the plates where there is no antibiotic.
If you have any questions at all our specialists are waiting to help you. Simply contact sales@intactgenomics for assistance.
Transformation Troubleshooting. Resources/troubleshooting/transformations. (n.d.). Retrieved January 14, 2022, from http://2018.igem.org/Resources/Troubleshooting/Transformations