Need to add 12 bp at the beginning of the sequence - (Feb/22/2016 )

I have DNA sequence to which I want to add 12 bp sequence starting at the position 16 bp. Is it possible to make this insertion using PCR? If I design primer that is 27 bp long (15bp matching the beginning of the sequence +12bp insert), do you think it will that work? Any other suggestions? My DNA sequence is 1200 bp long and the reverse primer will be designed appropriately.

Absolutely this will work by PCR. Don't worry about the length of the primers, just use the approximately 20bp that you would regularly use for a PCR, and add the additional bases to the 5' end of the primer. The additional bases do not count towards the annealing temperature of the PCR as they are non-specific, but will get incorporated after the first few rounds of the PCR cycle.

bob1 on Mon Feb 22 22:49:18 2016 said:

Absolutely this will work by PCR. Don't worry about the length of the primers, just use the approximately 20bp that you would regularly use for a PCR, and add the additional bases to the 5' end of the primer. The additional bases do not count towards the annealing temperature of the PCR as they are non-specific, but will get incorporated after the first few rounds of the PCR cycle.

I actually want the extra stuff to be inside of the sequence. Let's say I have a sequence 5' NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN....3' and want to insert XXXXXXXXXXXX with PCR, so that I have 5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNN....3'

So basically my question was how can I insert the sequence X with PCR? Will it work if my primer is 5' NNNNNNNNNNNNNNNXXXXXXXXXXXX?

No, that won't work. Your primer has to anneal well at the 3' end, and this will not anneal at all at the 3' end. You'll need to make a primer

5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNN....3'

There should be no problem making this primer.

Thanks everyone for their help! I used 5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNN primer and it worked, the 12 bp region was inserted in the right place :)