I have to amplify from a clone or from cDNA so I need the atg and the sequence in capitals to be amplifed upto TGA stop
I need to add SpeI at 5'(actagt) and SalI at 3' (gtcgac) for ligation of the product. both need 0bp extra for cut however I have added 1bp to be sure and digestion in compatible buffer is 75% effiency so plan to do a long digest overnight.
Basically I want to know do I design just on the section that will anneal or do I need to include the resitions sites that I am adding as well to design.
I tried primer3 but keep getting "High end self complementarity" does this matter much
here is part of the sequence just in case anyone can see an obvious primer combo
Can anyone help?
Edited by qnc, 13 October 2004 - 08:12 AM.