i'm trying to clone my gene in pet16b to further make histagg protein purification. Where can I do the cloning for a successful cloning and which restriction enzymes to use? I also want to get a soluble protein ,how can I process?
Hello the following sites are available in pet16b MCS: ndeI,xhoI,bamHI. When you clone in this MCS your protein will be tagged with 10X his tag that is a part of the vector. Also I have attached a map of pet16b that can help you with your cloning (you can use snapgene viewer or other software to view the file).
Dear Abire
on my blog https://proteocool.blogspot.com/
at ProteoCool n2: Primer desing fpr restiction/ligation cloning.
in case yoy are interested i suggest to you to evaluate also the use of PIPE cloning which is a flexible, powerfull and simple enzyme Free cloning approach ( ProteoCool n°1 and ProteoCool n°3 on the same blog)
however here you can find hints for select the right enzymes but of course the solubility of your target depends from its properties in terms of aminoacid sequence and from the domain that you would like to clone. you can find some more information about domain desing on the attached file which is also available in my research gate profile.
good luck
Manuele
can someone send me a link where I can find a good detailed explanation of how restrictions enzymes work when doing ligation?
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