Hello,
I have used RF-cloning successfully several times. Recently I have attempted to substitute 8 aa using the technique. I have made two sets of primers that are slightly different. Both sets have not worked. I have also eliminated the 55C annealing temperature and gone on with the 2 step protocol (68C annealing and elongation). My two project files are belo .rf-cloning.org/rf_cloning_pr … becb9fc7a4, rf-cloning.org/rf_cloning_pr … c1a0bcb569, Is this because of poor primer design?
Please help
I didn't find the right solution from the internet.
References:
www.rf-cloning.org/.../viewtopic.php
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Thank you