Hello everyone,
I faced a problem during my gene knockout experience, as is shown in (figure 3) I designed two primers for my targeted gene, then in (figure 4), I digested my targeted gene and inserted the cat gene into it, then by using the electroporation method I transfer the DNA which has the cat gene into the wild type (ZM1, ZM2) strains.
In (figure 1), I did PCR for the transformant strains, but as it is shown, there is not a good result from my PCR, and (figure 2) is the PCR of my targeted gene without inserting the cat gene, and I used the same PCR condition for both, and there is a result.
My question is how do I know that the DNA is inserted into the strains and the targeted gene is already knocked out?
Your suggestions will be highly appreciated.
You can try increasing the extension time in your PCR cycle. If you are not getting the 4 Kb amplicon using 1800F and 1800R you can use diferrent primer combinations mentioned below and the chimeric amplicons should be below 4 kb size (you can predict the exact size from the sequence that you might already have).
You can design primers that bind within the cat gene and run PCR using 1800F with a reverse primer within cat gene sequence and 1800R with forward primer within cat gene. If you obtain specific sized amplicons, the chimera amplicon will be indicative of successful insertion. Please refer to the image I have drawn. You have not shown the directions of your primers, so I have assumed the directions and drawn accordingly. If the directions of your primer are as shown in the drawing attached, you could also use 1800F and catF primer pair or 1800R and catR primer pair.
You really do not have good PCR products showing in Fig 2, so it is not going to be possible to determine whether you have the right strains or the right integrants. You might need to work on your PCR conditions or perhaps generate better template.
Thank you so much Dr. Michael J. Benedik, Dr.Niharika Ghoghari. The picture below is the result of the nanodrop, and also the PCR condition. the master mix that I created was:
1. dNTP 5ul
2. buffer 5ul
3. Taq polymerase 0.5 ul
4. (F, R) primer 1ul each
5. DNA 1ul
6. distilled water 36.5
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