I was trying to generate transgenic plants harbouring CRISPR/dCas9 construct. My plant selection marker is hygromycin. I have assayed the T0 plants for the presence of the hygromycin gene and all of them gave the desired sized bands upon PCR for amplifying the hygromycin gene. However, when I tried to amplify the dCas9 gene, no bands were observed. I have repeated this many times but no bands were observed. Can anybody please help?
It might be a primer issue. Try using your diluted plasmid as a positive control.
I have tried doing PCR with both genomic DNA and cDNA. In both the cases I have not seen any band for dCas9 whereas the hpt band was prominent.
So, it sounds like your gDNA is good as is your cDNA. Try your dCas9 primers with diluted plasmid to make sure they work then re-try your PCR.
Ok, then try to amplify the fragment between hpt and cas9 by using gDNA and anyone primer from both the genes .
Controls:
1) Negative Control: Check if the primers used to amplify a fragment of the Hygromycin also amplify something from non-transformed (WT) template. Ideally, they should not.
2) Positive Control: use your original CRISPR/Cas plasmid as a positive control in your PCR. Your dCas primers should give a band.
You start wondering/optimizing other factors only after these two controls are as they should be!
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