Maximum size of overhangs in PCR - (Apr/26/2011 )

I'd like to add some DNA to the ends of my amplicon, but what are reasonable limits; are there any? Overlap-extension PCR suggests that there must not be but has anyone information to the contrary?

Hi Sean,
I don't understand what you intend to do, do you mean add a few bases more in front of your desired fragment?

adrian kohsf on Wed Apr 27 02:28:43 2011 said:

I would really like to amplify a fragment and add to it LoxP sites (34 bases), followed by multiple cloning sites (say, another 30-40 bases). This would be similar for both primers. Is a 70-80 bp primer overhang acceptable?

Yes, it is possible, but you have to be careful of hairpin structures and unintentional priming of your template with the primer and primer-dimers. In other words, you need to be even more careful than usual in the design of the sequence.

phage434 on Wed Apr 27 11:47:31 2011 said:

LoxP sites are complementary to themselves, so will form hairpins and primer-dimers Foiled again... I'll have to find another way to get them in place. Thanks for the warning.

The regions of concern are ones that leave a 3' end bound to either another portion of the oligo or to a region of the other primer. It's the 3' end that matters. Hairpins with the 5' end, or even ones that leave a few mismatched bases at the very 3' end are much less important. You can probably work with the loxP sites if they aren't at the 3' end.

I wouldn't do it, because primer above 50nt are extremely expensive (HPLC cleanup and stuff). And then they may not work. Better chop it down or clone it from other sources. Well, maybe you don't have financial restrictions, then you can of course just try. Usually, as has been mentioned, if the 3' end is ok, then the primer should be working, regardless of the 5' length or composition...