Hello everyone,
I designed primers for Gateway cloning, but I'm unsure about the sequences I need to add. I plan to transfer my genes along with their native promoters into a donor vector. I added GGGG-attB1 and 2 seq-gene to forward and reverse primers. According to the information I gathered, I need to make changes to the stop codon region related to the destination vector, which I will use for the LR reaction. My destination vector contains a GFP tag. In this situation, should I design my reverse primer starting from the stop codon-containing region and eliminate the stop codon? If you have any additional insights that might be important for this experiment, could you please share them?
Thank you...
https://www.biochen.org/tools
Use this online tool to make reverse primer, when you open the online application there is a option of reverse complementry, just past reverse primer cds sequence and then click on reverse complementry, your reverse primer ready
Hello Busra, I do not fully understand your cloning setup, but you can explore using RecA-independent recombination or RAIR cloning as an alternative. RAIR cloning is pretty straightforward, but becomes more difficult to achieve depending on the number of inserts one is trying to clone into the vector. Essentially, it is homology-directed recombination where you PCR amplify the vector and insert, mix each PCR reaction with heat shock cells, and do colony PCR the next day to check for successful clones. Most common bacterial strains used in labs are capable of performing RAIR, all you need do is buy primers. See the publication below. Good luck!
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