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Lecture 3

BIO372 - Lecture 3.pdf

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Gordon Anderson

Lecture 3 January-16-14 10:06 AM Primer Extension Sanger sequencing is an example of primer extension Description:SuperScript Reverse Transcriptase (RT) is a DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene from which the RNase H sequence has been deleted, reducing the RNase H activity. This structural modification prevents degradation of the RNA molecule during first-strand cDNA synthesis (Figure 1). Purified from E. coli expressing the pol gene of M-MLV (4,5), mutagenized to reduce the RNase H activity (6). Unit Description:One unit incorporates 1 nmol of deoxyribonucleotide into acid- precipitable material in 10 min. at 37°C using poly(A)•oligo(dT)12–18as template•primer (7). Unit Reaction C3ndition:50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl , 1 mM 2 DTT, 0.5 mM [ H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT) 12–18 0.1 mg/ml BSA, and enzyme in 50 µl for 10 min. at 37°C. Contents and Storage:SuperScript RNase H Reverse Transcriptase is supplied with a vial of 5X first-strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl ], 2 vial of 100 mM DTT. Store at -20°C. Reference(s):1. Gerard, G. (1989) Focus® 11: 66.2. Howland, P. et al. (1991) Mol. Brain Res. 11 : 345.3. Gerard, G.F. et al. (1993) in Methods in Molecular Biology, Vol 16: Enzymes of Molecular Biology (Burrell, M. Ed.), Humana Press, Totowa, N.J., p. 73.4. Kotewicz, M. et al. (1985) Gene 35 : 249.5. Kotewicz, M.L. et al. (1988) Nucleic Acids Res. 16 : 265.6. Houts, G. et al. (1979) J. Virol. : 519. This is only about 30 yrs old. It is very common now. This is only about 30 yrs old. It is very common now. Run-off transcription Takinga DNA templateand copy into RNA Gives idea of the size of RNA transcript So we can measure the size of the dam Two types: Double stranded and single stranded - we wantto measuresingle stranded nucleic acids Nuclear run-on transcription What processes involved in the level of transcript? RNA is being degraded. It is very sensitive to degradation. 2' position - the OH group is much more susceptibleto attacks We can always get rid of DNAses - put into hot water bath (around 65) but this temp will not kill RNAses. RNAses are robust to degradation We have to worry aboutsynthesis and degradation - how to separatethe two processes? 32P-GTP - An RNA precursor, must be labeled in alpha position 32P-GTP - An RNA precursor, must be labeled in alpha position Labeled, then wash it with unlabeled to preventincorporation - a Pulse chase experiment The cat reporter gene to measure transcription Cat - enzyme that deals with chloramphenacol Like electrophoresisbut only looking at thin layer of solvent There are different acetylatedforms - can be based on degrees of acetylation Other reporter genes: GFP - green fluoresence protein lacZ Beta-galactosidase Accumulation of acetylated CAM
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