Project protocol - Contents
- Workflow and sampling
- Equipment and supplies
- Reagents and solutions
- Procedure: PCR and sequencing
- Definitions and calculations
Step Procedure performed Data collected 1 PCR of Gpr84 exon 2 - 2 Purification of PCR products - 3 Sequencing of PCR products Gpr84 exon 2 sequence
- PCR System
- QIAquick PCR Purification columns (Qiagen, Valencia CA)
- ABI 3130XL DNA sequencer (Perkin Elmer)
- Gpr84 exon 2 primers (sequences below)
- AmpliTaq polymerase
- PCR buffer 10X (Applied Biosystems, Foster City CA)
- ABI-PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer)
Gpr84 exon 2 primers. FOR:GCAAGTTCTCATACCATCTCCC; REV:AGCCCAAGCACAAAGTAGATG
- Reactions are prepared to contain genomic DNA (50ng), 5 pmol of each primer, 1.5 mmol/L MgCl2, 0.2 mmol/L of each deoxyribonucleotide, 2 µL PCR buffer, and 1.5 units of AmpliTaq DNA polymerase.
- Samples are subjected to 1 cycle of denaturation (95°C, 60 s), followed by 35 cycles of denaturation (95°C, 35 s), annealing (58°C, 45 s), and extension (68°C, 45 s).
- PCR products are purified with QIAquick PCR Purification columns.
- Purified PCR products are sequenced using the ABI-PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit.
- Samples are run on an ABI DNA sequencer.
- Gpr84 = G protein-coupled receptor 84
- Gpr84 allele designation:
- 0 = 2bp deletion (framshift, premature stop codon)
- 1 = no deletion (full length protein)
- Gpr84 exon 2 sequence