Hello everyone,
I have been having problems with insertion of my gene (195bp long coding sequence) into pGBKT7 using Gateway technology. I always perform LR reaction (incubating for at least 1 hour but I also tried overnight), then transformation of competent cells (One Shot™ TOP10 Chemically Competent E. coli), PCR screening, elfo, choose what looks promising on the gel , isolate pDNA and send it for sequencing. My gene is present in the vector (full length) but always in the opposite direction (3´-5´)...I am getting really desperate because no one has been able to help me with this so far and I need to proceed with my work as soon as possible. . Anyone experienced something similar or have an idea how to solve this ?
Dear Kristina
just a question: Did you perform LR reaction using which entry vector? Because in my experience the reverse insertion is a drawbacks of the topo reaction and not the lr. Check your entry vector because if the problem is there you need to go back.
however for the future i suggest to you to evaluate PIPE cloning, before discover it, i also was a fun of topo cloning and Gateway but it really changed my perspective.
if you are interested to known more details about PIPE you can find sone video tutorial on page 1 of my blog ProteoCool https://proteocool.blogspot.com
good luck
Manuele
Hello,
I used pDONR™221 as an donor vector to generate entry clone. Also, it's probably important to mention that I also worked with another 3 vectors : pGADT7 AD (which I plan to use for Y2H together with pGBKT7 BD), pE3130 and pE3136 (those for BiFC). I was successful with these 3 vectors and obtained positive colonies - i.e. gene was in the correct direction. So pGBKT7 BD is the one I still struggle with.
And may I ask what exactly do you mean by checking the vector ? I am still just an undergraduate student so I may not exactly understand since I dont have much experience.
And thank you for your advice!
i mean are you sure that the srquenve of the entry vector is it correct and the gene is not in rhe wrong direction also in this vector? Did you checked the sequence of the pdonr221 clones?
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