poor amplification with fusion primers

[Seqasaurus]

Hi

we're having trouble with some of our barcoded 16s primers. We are using 27f(fused with primer B) and 519r (fused with MID and primer A). These exact primers have been used before by others (Wu et al 2010) with much success.

We have 16 different barcoded reverse primers (with MIDs 1,2,3,4,5,6,7,8,10,11,13,14,15,16,17,18) and use the same forward primer for each. Unfortunately, primers with MIDs 1,2,7,8&16 do not amplify and MIDS 5,14 & 15 amplify very poorly. At first I thought it was a problem with the cognate samples rather than the barcoded primers but the samples yield full-length 16S amplicons with 27f (fusion primer) and 1492r. Even in the same PCR run as the 27f/519r. Also the dodgy MIDs are equally rubbish at amplifying E. coli DNA. Does anyone have experience of dodgy barcoded primers? They were pretty expensive...

Cheers,
Paul

[Xterra]

Hi,

Would you please provide me with a link to the paper (Wu et al 2010) you are referring to? I would like to read the experimental part and then see whether I am experiencing the same problem you are describing here.
Thanks,

X

[mydogphy]

i also met the same problem and i got 40 MIDs fusion primer for amplification. ANyone got the idea to solve the problems?

[Strejda]

We have used many fusion primers both for 16s and functional genes. The majority of problems were actually connected to type of polymerase rather than the primers themselfs. So now we do sort of primer-polymerase optimalization.

Common problems are either no products or multipleproducts. We also experienced contamination in brand new polymerase with DNA, probably originating from a production strain. Lately these problems ocur more often than 2 years ago.