Creating a Gene Knockout Mutant.
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- Опубліковано 9 лют 2025
- This is a compilation of three short video parts, condensed into one longer video.
PART1: PCR primer design.
PART2: Gel electrophoresis and transformation of PCR product.
PART3: Confirmation of PCR construct uptake by the bacterial species under investigation.
The video covers all of the Primer design, DNA transformation and Homologous Recobination, gene knockout, DNA sequencing and antibiotic resistance.
Some of the methods outlined use Bioinformatics and PCR techniques for biotechnology.
I hope this is useful to anyone trying these sorts of things out for themselves.
Great video
For the forward primer complimentary sequence should be taken
Yes, the forward primers are complementary to the antisense DNA strand.
Well described video
Great! Glad you thought it was well described. Best wishes!
Do we need to add full gene sequence of kanamycin or portion of it
Yes, it depends on what you are doing. The example in the video is for a full gene knockout / replacement with a knamycin resistance gene. If the knockout is successful, mutants can be selected on media supplemented with kanamycin. Other methods may rely on specific gene disruption by inserting sequences or deleting parts of coding sequences. Best wishes.
@genomeprojects and is it necessary to put promoter also
In the video, the kanamycin resistance gene is expressed using a promoter already in the genome at the knockout site. You could probably use an existing promoter or insert one.