1. Chapter 28 Manipulating DNA

2. Chapter Objectives Know how the techniques of molecular biology work Understand how to use the tools of molecular biology Put it all together with a molecular biology project (we will work on this in class after we know the tools and techniques)

3. Techniques Restriction Enzymes Electrophoresis Hybridization probes DNA sequencing – Chain terminator – Automated – Emulsion PCR

4. Restriction Enzymes https://www.neb.com/products/restriction- endonucleases https://www.neb.com/products/restriction- endonucleases App: Search NEB tools in either play store or App store Remember overhangs, this will be important

5. Electrophoresis Polyacrylamide Agarose

6. Hybridization Probes Northern Blot – RNA detection Southern Blot – DNA detection Western Blot – Protein detection

7. DNA Sequencing Chemical Sequencing Chain Terminator (dideoxy nucleotides) – Automated (color) Emulsion

8. PCR 95 denature 55? annealing 72 elongate repeat

9. Tools RT-PCR Microarray Cloning – Plasmids – Bacteriaphage – Cosmids – BAC/YAC Site-directed mutagenesis Knockout mice

10. Real-time (kind of) PCR (qPCR) (sort of) Can quantify the exact about of mRNA using PCR Combine dsDNA binding dye with PCR

11. Microarrays

12. Cloning Plasmid – small, circular DNA – can hold about 10 kb Bacteriophage – larger, circular DNA – can hold about 20 kb Cosmid – combination of a plasmid and phage, can hold 40-50 kb BAC/YAC – bacterial artificial chromosome/yeast AC – can hold up to 500 kb

13. Site Directed Mutagenesis Change DNA in plasmid, etc. to make a mutation in protein (substitution/deletion) Can change any residue to any other residue – Implications?

14. Mice Knockout – Deletion of gene Knockin – Insertion of gene Genetic background – models

15. Active Learning Projects – Download serial cloner http://serialbasics.free.fr/Serial_Cloner.html – Build plasmids (learn about functions) – Create (on paper) a mutant protein Create an experiment to study your protein – Learn about creating knockout mice.