title: RIPCM H. Pylori 3.2K 1576 v1.0
status: Public on Nov 30 2010
contact.city: Moscow
contact.first: Kuvat
contact.last: Momynaliev
contact.email: dhoroshun@gmail.com
contact.address: Malaya Pirogovskaya 1A
contact.country: Russia
contact.institute: Research Institute for Phys-Chim Medicine
organism: Helicobacter pylori 26695
manufacture_protocol: Primers set for PCR were purchased from Eurogentec. 3242 primers were designed and synthesized to amplify the complete ORFs in the H. pylori genome of strain 26695. The primers were designed to amplify each ORF beginning at the start codon and ending at the stop codon. The PCR reactions were performed using 96-well plates in a 100 mkl reaction volume in an MJ Research (Cambridge, MA) PTC-225 DNA Engine Tetrad thermal cycler under the following conditions: 94°C 5 min, followed by 30 rounds of 30 seconds at 94°C, 30 seconds at 500C, and 4 min at 72°C, followed by one round of 10 min at 72°C. To control the yield and the specificity of the amplified ORFs, all PCR reactions were analyzed by electrophoresis on a 1% agarose gel. The 1576 PCR products that were successfully obtained (97%) were spotted robotically (Qpix, Genetix, England) in duplicate onto positively charged nylon membrane (Amersham) of 12X8 cm. After spotting, the nucleic acid samples were alkali denatured and fixed by baking the membrane at 80°C for 60 min.