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Lecture 2

BIO372 - Lecture 2.pdf

7 Pages
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Department
Biology
Course Code
BIO372H5
Professor
Gordon Anderson

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Lecture 2 January-13-14 10:48 PM GTAACTATCATGC-32P AGGTCATTGATAGTACGGTAA.. GTAACTGTCATGC-32P AGGTCATTGACAGTACGGTAA.. SNP = Single nucleotide polymorphism Haploids vary based on SNP position S1 nuclease mapping of the 5’ end of a transcript. An old method but still used. There is an easier method now. How to find start and stop signals - find mRNA and sequence it, then align with reference genome Label the 5' end of green segment Figure how far is transcription start site Get rid of label in blue strand 32P on gamma ATP - alpha beta gamma, why in gamma? Gamma is on N and that is transferred in the polynucleotide SI nuclease - Degrade single strand but not double strand All the enzymes were needed to be made in the old days, so this experiment took so long to finish But now, you can just order from biotech companies Description: Calf Intestinal Alkaline Phosphatase (CIAP) is a phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA. Applications: Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation (1). Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase. Source: Purified from calf intestinal mucosa. Performance and Quality Testing: Endodeoxyribonuclease, 3´ exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay. Unit Definition: One unit hydrolyzes 1 µmol of 4-nitrophenyl phosphate in 1 min. at 37° C. Unit Reaction Conditions: 1 M diethanolamine buffer, 10 mM 4-nitrophenyl phosphate, 0.25 mM MgCl (pH 2.8) in 900 µl for 10 min. at 37°C. Contents and Storage:Calf Intestinal Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [500 mM Tris-HCl (pH 8.5), 1 mM EDTA], vial of dilution dephosphorylation buffer [500 mM Tris-HCl (pH 8.5), 1 mM EDTA], vial of dilution buffer [50% glycerol, 25 mM Tris-HCl (pH 7.6), 1 mM MgCl ,2and 0.1 mM ZnCl ]. 2 Store Calf Intestinal Alkaline Phosphatase at -20°C. Reference(s):1. Schmidt, B. et al. (1998) Focus 0: 52. Description: Bacterial Alkaline Phosphatase (BAP) removes 3´ and 5´ phosphates from DNA and RNA. BAP is active at 65°C for at least 1 h and is inactivated by phenol extraction. - very hard to activate compared to CIAP Applications: Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation. Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase. Source: Purified from E. coli C90. Performance and Quality Testing: Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; dephosphorylationefficiency determined. Unit Definition: One unit hydrolyzes 1 nmol of ATP in 30 min. at 37°C. Unit Reaction Conditions: 10 mM Tris-HCl (pH 8.0), 1.5 mM [- P]ATP, and enzyme in 50 µl for 30 min. at 37°C. Contents and Storage:Bacterial Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [100 mM Tris-HCl (pH 8.0)]. Store at -20°C. Description:T4 Polynucleotide Kinase catalyzes the transfer of the -phosphate of ATP to the 5´ hydroxyl terminus of DNA or RNA.A T4 Polynucleotide Kinase Technical Bulletin is available. Applications:5´ end-labeling of DNA or RNA (1). Phosphorylation of synthetic lin
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