I am carrying out a CRISPR knockout using PX458 plasmid with the following information
GROWTH IN BACTERIA
- Ampicillin, 100 μg/mLBacterial Resistance(s)
- 37°CGrowth Temperature
- Stbl3Growth Strain(s)
My question: After cloning in the sgRNA into this plasmid, can I transform the plasmid using competent DH5 alpha considering the fact that the growth in bacteria is Stbl3 growth strain?
if your plasmid has no large repeat (like LTR from retrovirus) you can certainly transform dh5... the stbl are used for unstable plasmid that can recombine... just check that after purification your plasmid is still the same...
It is generally recommended to use the same bacterial strain for transformation as the one used for plasmid growth and amplification. This is because different bacterial strains can have different plasmid replication and maintenance mechanisms, which can affect the stability and yield of the plasmid.
In your case, the PX458 plasmid has been designed for growth in the Stbl3 bacterial strain, and it is likely that this strain has the necessary machinery for replicating and maintaining the plasmid. Therefore, it is generally recommended to use the Stbl3 strain for transformation as well.
That being said, it is possible to transform the PX458 plasmid into other bacterial strains, including DH5 alpha, using competent cells. Competent cells are bacteria that have been specially prepared to facilitate the uptake of plasmid DNA. However, the efficiency of transformation may vary depending on the specific bacterial strain and the type of competent cells used.
If you do choose to use DH5 alpha competent cells for transformation, it is important to carefully follow the manufacturer's instructions and optimize the transformation conditions as needed to ensure the highest efficiency and yield. You may also want to consider performing a control transformation using a known plasmid to ensure that the competent cells are functioning properly and to establish a baseline for comparison.
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