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Maximum size of overhangs in PCR


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6 replies to this topic

#1 seanspotatobusiness

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Posted 26 April 2011 - 03:01 PM

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?

#2 Adrian K

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Posted 26 April 2011 - 06:28 PM

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?
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#3 seanspotatobusiness

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Posted 27 April 2011 - 01:19 AM

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?


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?

#4 phage434

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Posted 27 April 2011 - 03:47 AM

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.

#5 seanspotatobusiness

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Posted 27 April 2011 - 08:50 AM

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.


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.

#6 phage434

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Posted 27 April 2011 - 12:09 PM

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.

#7 Rsm

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Posted 28 April 2011 - 12:50 AM

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...

Edited by Rsm, 28 April 2011 - 12:58 AM.

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