Hello! I would like to clone a very large fragment of DNA into my vector (which will be digested with restriction enzymes) . I will need to amplify this fragment by PCR in pieces (2 or 3) and then clone those pieces into the vector.
By designing the primers for amplification, how can i be sure that each piece will be cloned in frame with the following peace and avoid mistakes or additional bases between one piece and the other?
thank you
It's easiest to design this using automated software, like NEBuilder. You give it the sequences that you want, and it spits out the primers needed to put them together. (However, as an exercise, if you're doing it the first time, it is valuable to draw the entire sequence out double-stranded on a white board and try to comprehend exactly which base pair goes where). Finally I would recommend to break the reaction up into individual 2-part assemblies. This makes it easier to troubleshoot and learn from the experience.
At any rate, you should verify the final construct by sequencing, whatever the construction strategy. You don't want to take the chance that any mistake arising by PCR amplilfication might affect the results obtained with the clone.
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