How many of us request for PAGE purification of primers more than 30 mers in len - PAGE purified primers (Feb/26/2007 )

Dear All,

I've recently encountered a problem with my cloning, which was due to error in the primer sequence. There was a base jump, i.e. NNXNN becomes X-less (NNNN). I enquired about this and the company that synthesized the primer said that it's a normal occurence everywhere. The company was kind enough to provide me with the error rate in primer synthesis:

Length (no. of bases)-----------Product (%)---------------Failure (%)
-----------10-------------------------91---------------------------9
-----------20-------------------------83---------------------------17
-----------50-------------------------61---------------------------39
-----------75-------------------------48---------------------------52
-----------100------------------------37---------------------------63

I would like to know your opinion regarding PAGE purification of primers. Is it neccessary to PAGE purify after 30 mer? I do PCR tagging regularly and the primers I normally needed are in the 60 to 70 mer region. If the information above is accurate, I'm actually gambling (50-50). Ha!

Bests

Short fragments (missing 5' end) are the most common failure, but are usually less of a problem experimentally. Deletions, as you noticed, are the next most common problem and show up a lot. I presume you are talking about picking a clone and seeing the deletion, as opposed to all of your primers having the deletion (which can also happen due to computer or mechanical error) but is much rarer.

I never gel purify. I think there is a large difference in quality among manufacturers on this point, but none are perfect, to be sure, as the process itself is error prone. With a 30-mer your chances should be much better than 50-50 for deletions, although there will likely be lots of 5' shortened fragments, perhaps 50%.

The failure rate depends upon the efficiency of the capping reaction during synthesis. A good run at 99.4% will generate 83.5% full-length (FL) 30 mer. 99.2% = 79% FL, 99.0% = 74% FL, 98.5% = 64% FL, 98.0% = 55% FL, and 96% = 29% FL 30 mer Basically, you get what you pay for. Note that even at 99.4% efficiency, 16.5% of the primers will be missing a base. For this reason, it is always best to pick more than one clone after transformation, since you will have to screen several to find one without a deletion in one of the primer regions.

We routinely have all of our oligos HPLC-purified, and only rarely resort to gel-purification of 80-90 mers.

Hi,

Many thanks for the info and suggestion.
It's not cheap having the primers purified each time it exceed 30 to 40 mer. I guess it's ok then just to pick more colonies to screen for the right ones.

I'm still having doubts deciding if purification is neccessary when primers exceed 60 mers for cases such as PCR-tagging. Has anyone tried using primers w/o purification when their primers exceed 60 mer? Is it a norm to do so in your lab (with minimal problems)? Appreciate your feedback.


Cheers

i cloned a 80bp sequence in just desalted purif level, ordered at sigma.

Thanks fred_33.