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#1 2015-12-21 06:00:15

Registered: 2015-12-21
Posts: 1

to design an RF cloning project with the website

"From the home page, paste your destination plasmid sequence into the upper text area, and your desired insert sequence in the lower text area. You can also select from popular plasmid sequences, or any sequences you have saved to the database from the associated pull-down menu. Indicate the insertion points within the plasmid, either by adding two exclamation "!" points directly into the sequence, or by specifying the positions in the appropriate text boxes. Click run, and the software will generate custom hybrid primers to accomplish your cloning project. You can further manipulate the primers by shifting the insertion points, or by increasing/decreasing the length of the sequences complementary to the plasmid or insert, using the provided arrow buttons. Annealing temperatures are calculated for the primary and secondary PCR reactions, the expected length of the primary PCR product is given, as well as recommendations for the secondary PCR conditions. It has been my experience that the high-fidelity long-read polymerases iProof and Phusion (available from Bio-Rad and NEB respectively) produce the best results, and the extension times calculated are based on these enzymes. Other high fidelity polymerases can also work, although the manufacturer's instructions should be followed to determine appropriate PCR conditions." here is some suggestion about the RF cloning project, I think it is very helpful in this field. so I want to share with all of you.

Last edited by mandyscott (2015-12-21 06:00:50)


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