Please suggest me what should be done in case I'm doing random integration of my protein localization construct in a fungus by means of ATMT (Agrobacterium Mediated Transformation) where I don't want a site specific integration. Is Southern blotting is essential to show the confirmation of the positive transformation or simply genomic PCR is sufficient for confirmation? Please help and reply asap!
The main concern I see with this is that PCRs can be incredibly sensitive -- so you can't necessarily distinguish between a non-integrated contaminant (plasmid, PCR product...) that fell into the tube from true integration. It can be difficult to work cleanly enough to rule that out, so you can't take it on faith. You'll have to test it quantitatively.
What I would do is a copy number control. You'll need a quantitative method, that can measure sequence copy number. Real-time PCR would be ideal, but you can approximate it by running gels too, trying to catch the reaction before the band intensity is topped out (cycle ~10-20?).
If your construct copy number equals the genome copy number (measured by a genomic control gene), then you can be reasonably confident that you're looking at integration. Whereas if you have many more genome copies than construct, then it's likely that you're really measuring a non-integrated construct contaminating your genomic prep.
yes, if u do a qPCR and show data for copy number, you will not need to re confirm by southern
good luck
Can you please tell me in detail how to check the copy number in real time PCR, I shall be very thankful to you.
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs