Restriction enzyme digestion and ligation protocol control,digestive enzymes de 4life wikipedia,garden of life baby probiotics thrush,can probiotics cause liver pain nausea - Plans Download

It is ideal that the insert is cut with BioBrick official restriction enzymes (requirement 2) however, cloning site must be cut without using those enzymes (requirement 3). We designed two Bsa I site to produce Not I like overhang and Spe I like overhang respectively. If this goes on, we have a serious problem that the final product has Xba I site after the Not I site (fail to fulfill the requirement 3). To solve this problem, it is easy to add only a Not I site and a Spe I site at each end of the insert part. In this construct, the inserted GFP is interchangeable with another BioBrick part using Bsa I again, you can easily identify the replacement of the insert by the color of colonies.

The FB Plasmid Miniprep Kit provides a fast, simple, and cost-effective method for isolation of plasmid DNA from cultured Gram positive and Gram negative bacterial cells. Overhangs resulting from Bsa 1 digestion should not be the same to prevent ligation in incorrect direction(requirement 4). Bsa I recognition site and its cutting site are separated, and the sequence on cutting site can be changed to suit different needs. What is worse is that an in-frame stop codon appears on the Xba I site, just before the insert.
However, it requires different primer set that anneals each insert part (fail to fulfill the requirement 2).

This primer causes a point mutation to delete the Xba I site and its internal in-frame stop codon. Its design is based on alkaline lysis of bacterial cells followed by binding of DNA onto the glass fiber matrix of the spin column in the presence of highly concentrated salts.
Using the "Xba_byebye" prefix primer and the normal suffix primer, we can fulfill all the requirements.

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