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-   -   tdTomato cloning destroying life! (http://www.molecularstation.com/forum/molecular-cloning-forum/70892-tdtomato-cloning-destroying-life.html)

fiz1999 10-13-2009 01:00 AM

tdTomato cloning destroying life!
 
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!

-fiz1999

AdamP 03-09-2010 03:44 PM

Re: tdTomato cloning destroying life!
 
Hi

I hope you have sorted out your problems by now. I have used tdTomato and not had any problems. I also ran a PCR and gel purified out the upper band and then digested it, so the gel purification won't have affected the restriction sites. Did you leave enough bases at the 5' end of your primers for recognition by the restriction enzymes?

scienceguy 03-09-2010 05:17 PM

Re: tdTomato cloning destroying life!
 
I am running into the exact same issue with TdTomato. I am recombineering so no need to cut anything but I am getting 2 bands, 1400 and a brighter primary product at 900. Tdtomato is a tandem dimer so I thought that the primers could be laying down in the middle and at the end, but I moved one primer out into the MCS and still get 2 bands. Let me know if you ever resolved this issue. If anyone can give me the primer sequences that they have used (at least the 3' ends) that would be great!

richardm@ 10-15-2010 03:40 PM

Re: tdTomato cloning destroying life!
 
Hey,
I'm having an identical isssue. Did anyone manage to resolve this problem? Whatever I do I seem to get a really strong 900bp band and a very weak 1400bp.
Kind regards
Richard

scienceguy 10-16-2010 04:21 PM

Re: tdTomato cloning destroying life!
 
Yeah, you need to slide one of your primers down a bit. Most of the tdtomato coding sequence repeats since it is designed as a tandem dimer. so your primer is laying down in the middle and at the end of the coding sequence. Pay close attention to the 3' end of your primer and make sure it is at a unique sequence. Good luck!

TGS 10-19-2010 08:54 PM

Re: tdTomato cloning destroying life!
 
It's not that unusual to get side-products when using PCR. I'd just cut out and clean the band which seems to have the right size and work with it. If one is unsure if it is the right amplificate one could always perform a test-digestion with the purified DNA.
Since I never worked with tdTomato I don't know the sequence and subsequently don't know the digestions sites, but there probably is the one or the other you could use?!

eristic 06-12-2012 06:26 PM

Re: tdTomato cloning destroying life!
 
I am trying to amplify tdTom to make a new enhancer tester vector but I am not able to get any bands. Can someone send me their primers that they have used successfully please?


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