I have to make full length cDNA clone how should i proceed for that as far as i know i have to design primers that will cover full length of cDNA if I am not wrong i tried to design primers but i couldnt as its not covering the whole lenght the length is 1456 can u plz suggest me how i can design primer so that it will cover the full length of cDNA SEQ.
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1 reply to this topic
Posted 12 January 2010 - 04:26 PM
My recommendation would be to try and design primers for 300-400 bp at a time, with some overlap (8-10nts) between primer sets. Amplify individual pieces with a high fidelity polymerase and simply multiplex the pieces together, again, using a proof reading thermopolymerase. Will make your life a lot easier than going for 1.4KB in 1 shot!