Patti continued on by explaining that there is a very delicate technique for hand-rolling the primer mixture
into the forms, so much so that the direction and hand pressure needs to remain constant on every stroke.
025 [micro]M primer mixture
containing 14 specific tag-labeled allele-specific primers for 7 common and 7 variant alleles (synthesized by Sigma-genosys); and 1.
5 U SuperScript II was added to the RNA primer mixture
4 [micro]L of 20X EraGen CFTR PCR primer mixture
, l x Titanium Taq, and 1 [micro]L of diluted genomic target DNA.
A MPCR reaction mixture was prepared containing 25 [micro]L of Qiagen MPCR master mixture, 5 [micro]L of 10x Tris-EDTA primer mixture
, 5 [micro]L from the E.
We added 1 [micro]L of a 1:100 dilution of PCR product from the multiplex round to 60 [micro]L of this buffer and then added 5 [micro]L of the DNA-buffer mixture to each well, which contained 10 [micro]L of mineral oil and 3 [micro]L of allele-specific primer mixture (see Table 2 in the online Data Supplement).
Each assay uses the same template DNA with only the primer mixture changing, facilitating ease of use.
The retention time for primer HBB3Um2 is the mean value obtained from the primer mixture
(the first injection in every batch of runs) because the amplitude of this particular primer peak was usually too small in the test samples to give a retention time reading.
To each tube of "purified" PCR product we added 1 [micro]L of primer mixture
(consisting of seven different mutation-specific primers), 2.
To each tube of purified PCR product, we added 1 [micro]L of panel A or B mutation-detection primer mixture
(see below), 2.
Less than one microliter of the purified, labeled primer mixture
was enough for a PCR reaction.
The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures