I'm performing Tol2 enhancer and promoter assay in Zebrafish and would need a help with the constructs. I've ordered few of them from Addgene and they are on their way (for promoter assay: p5E-MCS and pME-mCherry), but I desperately need destination vectors. I would need pGW-cfos GFP (for enhancer assay) and pTol 2 DestR 4-R2pA (for promoter assay) and pCS2FA-transposase (for co-injection of transposase mRNA). Does anyone have them?
Furthermore: do you have any idea how to clone my blunt PCR product into p5E-MCS (with attL4/R1 sites)? I don't want to redo the PCR as I'm trying to use the exact construct that my colleague has used in a previous cell culture assay.
Hi Monika,
For getting the destination plasmids, you might wish to try this link: http://tol2kit.genetics.utah.edu/index.php/Main_Page
It will have the pDEST and pCSF2A vectors but I'm unsure about the cfos GFP.
For cloning your blunt end product, you could digest the p5E-MCS with EcoRV which gives blunt ends and then ligate in a 1:1 molar ratio. Make sure you dephosphorylate your vector backbone to avoid getting too many re-ligations and also run a vector only ligation as a control.
Hope these are of help. If you need the pCSF2A urgently, let me know and I'll send it to you. Unfortunately, I have the pDEST R4R3 and not R2, so won't be much help there.
Goodluck with your experiments!
Navneet
Dear Navneet,
thank you for your answers! I was just wondering - will this not be to unstable if I perform the ligation with blunt edges? Or would it be easier if I make a TA cloning and cut with some other enzyme?
And on this note - how do you dephosphorylate the backbone :)
For the plasmids - I've got in contact with authors, so I'll be getting them soon. I decided to go with pCS-zT2TP for the transposase and I'm getting the R4-R3 destination as well :)
I really appreciate your help!
Best wishes from Scotland!
Monika
Hi Monika,
Blunt end ligations are less efficient in general but they do work if done properly. The key is to dephosphorylate the vector properly by using an alkaline phosphatase enzyme like this: https://www.neb.com/products/m0371-shrimp-alkaline-phosphatase-rsap
It will be much easier ofcourse if you can generate the PCR product with 3' A overhangs. This will allow you to ligate it into a plasmid like pCR2.1 or pGEM-T and then shuttle it from there into p5E-MCS.
Hope everything works well.
Navneet
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