The master sample was stratified by province, residence and race, and the EAs were then selected with a probability proportional to size, with the estimated number of people aged ≥50 years in each EA as a measure of size.
An example of a combination of sampling methods, a master sample design, is given to show that it usually takes some engineering to produce a good sample design.
From these master plates, a sample was replicated with a 96-well replicator to a microtitre plate containing YEPD and grown for 3 d.
A master mix for each sample was prepared and consisted of 1x Kapa Taq buffer (Sopachem), 1 mM MgCl2 (Roche Diagnostics), 0.12 mM dNTP's (Invitrogen), 0.02 U/μl Kapa Taq polymerase (Sopachem), 0.32x LC Green Plus (Bioké) and 25 ng gDNA per reaction.
In this process, the reads were aligned against 414 known subtype B HIV-1 genomes from the Los Alamos National Laboratories using the iterative mapping method described in the Methods section, and the master reference sequence for this sample was constructed by combining regions of consensus where the Illumina reads mapped to each of these genomes.
The master mix for each qPCR sample was prepared to a total volume of 5 µl for a 384 well-plate: 10 ng cDNA template, 0.55 µM forward primer, 0.55 µM reverse primer, SYBR green master premix (Takara Biotechnology Co Ltd, Dalian) and RNAse-free water (Ambion, Cat AM9937).
The master mix for each cDNA sample was composed of 5 μl of the diluted cDNA (20 μl cDNA + 80 μl of nuclease-free water Ambion Austin, TX), 15 μl H2O, 5 μl 10 ng/ml reverse and forward primers, and the 25 μl Q™SyBr Green Supermix (Bio-Rad Hercules, CA).
The human faeces sample were collected from master and graduate degree students residing at the institute hostels of National Dairy Research Institute (NDRI), Karnal, India.
1.25 µl of cDNA from each sample was mixed with master mix consisting of 5 µl Cells Direct 2X reaction mix (Invitrogen), 2.5 µl 0.2X PPP mix (48 genes, Taqman/Applied Biosystems Inc, Foster City, CA, USA, http://www.appliedbiosystems.com), 0.5 µl Platinum Taq (Invitrogen) and 0.75 µl TE Buffer.
Each sample was mixed with 24μl of master mix (SYBR® Green PCR Master Mix – Applied Biosystems®).
