Hello,
I injected the plasmid pCFJ150 together with the selection markers into strain EG4322 with the direct injection protocol. I was able to get unc-119 rescued worms with no fluorescent markers after heating shock 2hrs at 34C. However, I wasn’t able to confirm the insertion of the plasmid in ttTi5605 site with PCR. I wonder if it is possible that the insertion could happen in other random sites?
I suppose that it’s possible, but pretty unlikely. Whenever I’ve used pCFJ150 it’s gone into the correct site. So both your 5’ and 3’ junction PCRs and long range PCRs across the locus failed? It could be some sort of aberrant multicopy insertion that leads to failed PCR. I’d try to get additional lines. Usually ~80% of the inserts that I’ve made into the ttTi5605 site were single-copy, similar to what the Frokjaer-Jensen paper reported.
Is there a possibility that the plasmid was inserted in an inverse direction? My 5’ and 3’ junction PCRs failed, I haven’t tried the long range PCR yet.
Would be weird if it went in completely inversed, but some sort of complex rearrangement is possible. Do the LongAmp PCR to see what the locus looks like. If it’s Wt size, then the insert may be somewhere else in the genome. If you can’t amplify across the insertion, that may suggest a complex, multicopy insertion. Have you tried PCRing whatever you are trying to insert into ttTI5605 to see if what you cloned into pCFJ150 is in the strain?