Mol Biotech Ch4&6

Mol Biotech Ch4&6 - Molecular Biotechnology Reading...

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Molecular Biotechnology Reading Notes Chapter 4 Fig. 4.1: Outline of cloning with plasmids. Fig. 4.2 & 4.3: Restriction enzymes can create staggered or blunt ends. They cleave phophodiester bonds. Table 4.1: Define: -- recognition site (can be 4-8 bp long) -- 5’ and 3’ overhangs (extensions) -- palindromic sequence -- isoschizomers (schism – cut or break) -- compatible ends (isocaudomers) (caudal – tail or end) Fig. 4.5: What is a restriction map? Fig. 4.7: DNA ligase is needed to seal the sticky ends together. Fig. 4.9: So, using our review of restriction enzymes, you can see how an insert is ligated into a plasmid. What is alkaline phosphatase used for in this process? What does it prevent? Define: Competent cells; transformation. How are competent cells made? What is the transformation rate for E. coli competent cells? How does the ampR gene solve this low transformation rate? Another problem is absence of insert in the ligated plasmid, due to unwanted self-ligation of the vector. How is this solved with the pUC plasmid lacZ system? Which colony color indicates the insert is present? What is a “multiple cloning site” (MCS)? Why is an MCS desirable? Fig. 4.12 & 4.13: What is a library? Libraries are created by partial digests. These create overlaping fragments. By choosing the proper digestion time, one can create overlapping fragments of a specific size range. Fig. 4.14: Southern and northern blots work by immobilizing a complex mixture of DNA or RNA onto a nylon membrane, and then binding (hybridizing) the probe to a specific DNA or RNA on the membrane. The probe can be either RNA or DNA. The probe is often radioactively labeled so that it can be detected by autoradiography, which involves placing X-ray film on the nylon membrane.
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Fig. 4.17: A library of clones can be screened by colony hybridization. This is like a Southern, except there is no gel. Instead, the cells in the colonies are lysed and the DNA is bound to the membrane. Fig. 4.18: Alternatively, a library can be screened for the proteins it produces, rather than the presence of a DNA. The protein is bound to the nylon and then antibodies are added instead of a probe. What is the purposed of the primary and secondary antibodies? What does the secondary antibody comprise? Fig. 4.20: mRNA makes up only about 3% of the total RNA of the cell. What does the 97% comprise? A total RNA prep can be put onto an oligo-dT cellulose column. This is a kind of paste composed of tiny cellulose beads. Only the mRNA is retained—why? Fig. 4.21: Now that you have isolated your mRNA, you can make cDNA from it. Describe how this is done in this figure and which enzymes are used.
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