my primers are F:GCATCATATGatgaatgggaccgcaaaccc and R:GCATCTCGAGttagtctttctggtaaggccg,my fragment that I want to clone start by atgaatggga.......,and my vector is pet16b.i used ndeI and XhoI restrictions enzymes for digestion and the same to test the ligation efficiency but I always got self religated vector. does someone have an idea to help me to improve my ligation?i didn't make vector dephosphorylation as I used two enzymes.
Hello, after the ligation reaction you can check if the T4 ligase was able to ligate your DNA fragments. Here you can find information on how this is done:
http://www.thermoscientificbio.com/whats-new-t4-dna-ligase/
If this experiment shows you that you have a positive ligation reaction then you can rule out the possibility that your ligase failed. It might at least help you coming closer to solving your problem.
Good luck!
self ligation may suggest that one enzyme is not working leaving the same cut site at both ends of the vector. Have you tried cutting a vector sample with each enzyme separately just to make sure that the enzymes are both working?
Abire Sigbessia if that is the case I suggest you follow Paul Rutland suggestion since it does indicate that one of your enzymes is not working. Basically run single digest using both enzymes and run on a gel with the uncut plasmid as a control.
If you find that both enzymes cut then I see that the xho site is at 324 bases and the nde site is at 331 bases. They are quite close .
Xho ideally needs 4 or 5 bases outside the cut site for good cutting but nde1 cuts poorly with one or two spare bases but cuts well with 3 spare bases so instead of a double digest do a single digest as Hanna Alalam suggests cutting first with Xho and then add Nde which is less fussy about the number of spare bases it needs. They both cut in the same buffer ( neb 4 buffer or cutsmart buffer
infomation
https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Hello, after the ligation reaction you can check if the T4 ligase was able to ligate your DNA fragments. Here you can find information on how this is done:
http://www.thermoscientificbio.com/whats-new-t4-dna-ligase/
If this experiment shows you that you have a positive ligation reaction then you can rule out the possibility that your ligase failed. It might at least help you coming closer to solving your problem.
Good luck!
thanks to all, now I've test the enzymes and the work well, but I don't know why I'm still getting the recombinant vector. I've tested many ratios,1:3 1:4 1:6 but still don't find my fragment ligated.
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