http://www.protocol-online.org/biology-forums-2/posts/9061.html Set up restriction digestsfor your insert (or donor plasmid) and plasmid backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid … See more Now that you’ve cut your insert and vector, unfortunately you can’t just throw the digestion mixtures together. You need to isolate your insert and backbone from the enzymes used to digest them as well as any pieces cut out or … See more In the ligation step, you mix your purified, cut backbone and insert in a single tube allowing the compatible overhangs generated by restriction digestion to anneal to one … See more Once it looks like your ligation has worked, you will need to pick individual bacterial colonies and check them for successful ligation. Pick 3-10 … See more Transformyour ligation reaction into your bacterial strain of choice. Follow the manufacturer’s instructions for your competent cells. For most standard cloning, you can … See more
choosing restriction sites - Molecular Cloning - Protocol Online
http://www.protocol-online.org/biology-forums-2/posts/9061.html WebGiven its prevalence, the vast majority of plasmids used for DNA cloning and expression contain several popular restriction enzyme sites. You can easily move (subclone) any piece of DNA that already has restriction sites on either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning site. mfs property services markham
Multiple Cloning Site - an overview ScienceDirect Topics
WebA restriction enzyme is a DNA-cutting enzyme that recognizes specific sites in DNA. Many restriction enzymes make staggered cuts at or near their recognition sites, producing ends with a single-stranded overhang. If two … WebSep 25, 2024 · Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting. Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT … WebBy choosing restriction sites with compatible ends that destroy the recognition site when ligated to one another, parts can be combined together and the original flanking sites re-used for the next round of … mfs piping and industrial