A 384-well plate was first loaded with reagents manually, including a primer mixture
for each assay and a template mixture for each sample analyzed.
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.
Following the annealing of the random primers to the RNA, 20 [micro]L of first-strand reaction mix consisting of 2x first-strand buffer (Invitrogen Inc.), 10 mmol/L DTT, 500 [micro]mol/L deoxynucleoside triphosphates (dNTPs), and 7.5 U SuperScript II was added to the RNA primer mixture
. The RNA was reversed transcribed for 45 min at 45[degrees]C.
We applied 5 [micro]L of SAP- and EXO 1-treated PCR product as the template for ASPE in a total of 20 p.L of reaction mixture containing 1 x PCR reaction buffer; 1.25 mM Mg[Cl.sub.2]; 5 [micro]M each of dGTP, dATP, dTTP, and biotin-14-dCTP (Invitrogen); 0.025 [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 of Platinum GenoType Tsp DNA Polymerase (Invitrogen), or TITANIUM Taq DNA Polymerase (BD), or TaKaRa Tag[TM] HS DNA Polymerase (TAKARA BIO, INC.).
PN1235 or PN1305 for the 579T system; EraGen), 0.4 [micro]L of 20X EraGen CFTR PCR primer mixture, l x Titanium Taq, and 1 [micro]L of diluted genomic target DNA.
PN1310), and 0.4 [micro]L of 20x EraGen CFTR TSE primer mixture; final concentrations of TSEs were 25-100 nmol/L (see Table 3 in the online Data Supplement).
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).
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 panel A or B mutation-detection primer mixture
(see below), 2.5 [micro]L of HPLC-grade water, and 2.5 [micro]L of SnaPshot[TM] Multiplex Ready Reaction Mix (Applied Bio-systems) containing AmpliTaq[R] DNA polymerase and fluorescently labeled ddNTPs.
The sequence encompassing the MELAS mutation site was PCR-amplified with the Cy-5-labeled primer mixture
according to the conditions described by Goto et al.
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
. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B.