jsonp_1685114208201_84727({"objects":[{"commentText":"It's useful to study background of cloning","user":"soomin Lee","userUri":"","threadInfo":"0001.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1528990550748},{"commentText":"This is super useful and well done done, thank you !","user":"Quentin Giraud","userUri":"","threadInfo":"0002.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1592484920625},{"commentText":"Good job!","user":"Hero Godspower","userUri":"","threadInfo":"0003.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1601209893652},{"commentText":"Can you explain the point of the second diagnostic gel? Didn't we already verify the insert in the first gel since the insert is the PCR product","user":"Isha Kumar","userUri":"","threadInfo":"0004.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1612135519524},{"commentText":"Hi Isha, The reason you do the second gel is because the ligation reaction does not give you just the product you're looking for. For example, sometimes the vector backbone ligates back onto itself without the insert.\r\n\r\nJennifer","user":"Jennifer Tsang (Addgene)","userUri":"","threadInfo":"0004.0001.0000.0000.0000.0000.0000.0000.0000.0000","created":1612184867621},{"commentText":"Hi,\r\nthe insert we try to clone into a ~3 kb vector has a lenght of 13 kb.\r\nDoes it make any sense for the calculation of the ratio, to take the vector as insert and vice versa?\r\nGreetings","user":"Ralf","userUri":"","threadInfo":"0005.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1620045584589},{"commentText":"Hi Ralf,\r\n\r\nThanks for your question. The optimal ratio of vector:insert can range from 1:1 to 1:10 (although the higher amount of insert is typically used to insert multiple copies or for blunt end ligation). Since most protocols were developed for smaller inserts, it might help to try a range of ratios and include a no-insert control (ie, 1:0, 1:1, 1:3, 1:5).\r\nIt's better to have more insert available to ensure it forms the expected plasmid. If you have more vector, you risk it self-ligating and getting a final plasmid that is missing the insert.\r\n\r\n~Nyla","user":"Nyla Naim (Addgene)","userUri":"","threadInfo":"0005.0001.0000.0000.0000.0000.0000.0000.0000.0000","created":1620062868279},{"commentText":"Hi, what would you say are the main differences between restriction-based cloning and Gateway cloning?","user":"Isha Sachan","userUri":"","threadInfo":"0006.0000.0000.0000.0000.0000.0000.0000.0000.0000","created":1620141730771},{"commentText":"Hi Isha,\r\n\r\nGateway cloning doesn't rely on restriction sites or restriction enzymes. You can more information on Gateway cloning in our other blog post:  https://blog.addgene.org/plasmids-101-gateway-cloning\r\n\r\nJennifer","user":"Jennifer Tsang (Addgene)","userUri":"","threadInfo":"0006.0001.0000.0000.0000.0000.0000.0000.0000.0000","created":1620147283271}],"total":9,"limit":10000,"offset":0})