* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Pathogenic bacteria Genomic DNA extracted from
DNA barcoding wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Genome evolution wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Gene expression wikipedia , lookup
Gene regulatory network wikipedia , lookup
Gel electrophoresis wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular evolution wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA vaccination wikipedia , lookup
Silencer (genetics) wikipedia , lookup
DNA supercoil wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Molecular cloning wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Transformation (genetics) wikipedia , lookup
BIOL 4102 – Week 1 (Polymerase Chain Reaction) Week 1: Molecular Cloning of the SSB gene into the pCDF plasmid for protein expression. Introduction – SSB gene from a specific species of pathogenic bacterium has already been cloned into the pUC57 plasmid. SSB gene Pathogenic bacteria PCR using primers specific for the SSB gene was carried out to amplify SSB DNA Genomic DNA extracted from pathogenic bacteria SSB gene was inserted into the pUC57 plasmid by molecular cloning Ampilillin resistance gene pUC plasmid Ori SSB gene Week 1 – Learning the concept of PCR by amplifying E.coli SSB DNA from pUC57 plasmid. Overview: • To learn the concept and technique of PCR, SSB gene cloned into the pUC57 plasmid will be amplified using forward and reverse primers (shown in red). • The amplified SSB PCR product will be analyzed using agarose gel electrophoresis. Ampicillin resistance gene Ori pUC57 plasmid T7 SSB gene promoter T7 terminator PCR followed by agarose gel electrophoresis to analyze the DNA amplified by PCR. DNA size marker PCR product Gel Lanes 5 kb 2 kb 1 kb 0.5 kb SSB 0.2 kb Sofia Origanti and Edwin Antony, 2015 (Expected DNA size of SSB – 0.5kb) Week 1 – Learning the concept of PCR by amplifying DNA inserted into the pCDF plasmid. Overview (cont’d.) : PCR- Schematic Plasmid DNA + PCR reaction tube PCR machine PCR steps Cycle 1 Forward & Reverse + T7 primers Nucleotides + DNA polymerase 5’ 3’ 3’ SSB gene section of the plasmid 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Denaturation at 98°C separation of the two strands of double stranded DNA Annealing at 55°C –pairing of the forward and reverse primers (in red) with the complementary sequence on SSB DNA Elongation at 72°C –DNA polymerase (in blue) moves in the 3’ to 5’ direction of the template strand and adds nucleotides to the primers (in pink) in a complementary fashion 2 copies of SSB DNA at the end of first cycle Cycle 2 Exponential doubling with each cycle- 2 copies of SSB DNA become 4 at the end of second cycle. 4 copies become 8 with cycle 3 and so on. Sofia Origanti and Edwin Antony, 2015