By DONNA L. LILLIAN
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Extra resources for A thorn by any other name: sexist discourse as hate speech
6 + ) / ) / ) / 6 + 6 + 5 = Incorporation of nucleotides labeled with dGTP or dATP ✰ and unlabeled nucleotides • Synthesis incorporates the labeled nucleotides. • The length of the primers influences the length of the newly formed strands. 11 Labeling of DNA by random priming. ➫ Greater than or equal to 14,000 g ➫ Storage of products and precipitation of the probe ➫ Use carefully. Do not disperse the products on the wall of the tube. ➫ Denaturation of DNA ➫ Incubation of the enzyme REAGENTS • Dithiothreitol (DTT) • EDTA • Hexanucleotides ➫ Only used for in situ hybridization SOLUTIONS ➫ All the solutions are prepared using RNase-free reagents in a sterile container (see Appendix A1).
Probes are ligated to obtain smaller fragments. ➫ Construction of this is very important. ➫ Avoid all risk of contamination by RNases. ➫ Restriction enzymes have a limited lifespan and are very labile. ➫ The original probes are the same length as the DNA template. 2 Summary of Different Steps 2 3′ 5′ 1 3′ P2 P1 5′ cDNA 1 V EC OR T cRNA 5′ 3′ 2 P1 P2 Vector 3′ 5′ cDNA 5′ 3′ Cp1 3 5′ 3′ cDNA 5′ 3′ P2 Vector Cp2 cRNA 3′ 5′ 1 = Insertion into a plasmid • The target cDNA is inserted into a plasmid equipped with two promoters, P1 and P2, recognized by two different RNA polymerases and situated on either side and on each strand of the insert.
The insert is denatured to separate the two strands (by heat: 95°C for 5 min). ➫ The polynucleotides that serve as primers are generally hexanucleotides, but can be nonanucleotides in random sequences. ➫ The Klenow enzyme is the fragment of DNA polymerase I after digestion with subtilisin (disappearance of the DNase activity). This polymerase forms double-stranded DNA from a single-strand and a primer. ➫ The incorporation of the nucleotides carrying antigens is slower than native nucleotides.