Hi fellow scientists,
I have an issue with cloning tdTomato. I have been unable to clone this gene for a over month and am wits end here. Hope some experts out there can provide some advice.
I have designed appropriate PCR primers to amplify tdTomato. Unfortunately, the PCR product gives two bands. One band is about the about the correct size of the gene (~1400 bp) and the other is about half. I have cut out the larger band many times and tried to ligate it into my vector but to no avail.
I then thought that the restriction sites may be mutated on the oligos so I ordered new oligos with more overlapping sequence. Still got two bands of the same size as before. I also linearized the plasmid that I was amplifying off of because I heard that the pRSET B vector can coil easily. I consistently get the two bands and the larger band will not ligate into my desired vector.
Now I have changed the strategy and I am subcloning the PCR product into a TA cloning vector from invitrogen. I will digest out of the TA cloning vector into my final vector. However, I am still unsure if this is the correct PCR product.
I suppose I can also run a gradient PCR but I am surprised since many people are starting to use tdTomato for in vivo purposes. It all seems very hellish.
If anybody else has had this problem, please let me know. If you have a solution that would be even better. Thanks in advance everybody!