1. Lecture 6 Experiment 3: Basic subcloning Flowchart Experiment 4: Light regulated genes Principles and process of RNA isolation Assignment 1 due next week Discussion of Article 3

2. Experiment 3 Goal: To introduce basic procedures used to manipulate DNA. General procedures are DNA fragment isolation, ligation of DNA, transformation of bacteria, small scale DNA isolation.

3. Lab 6 DNA fragment isolation kit A Vector preparation B Fragment isolation

4. Lab 7 Ligation of vector and DNA fragment T4 DNA ligase

5. How do we ligate a XhoI and Sal I site together? GTCGAC SalI CTGCTG CTCGAG XhoI GAGCTC

6. Lab 8 A. Run ligation reactions on an agarose gel B. Transform bacteria

7. Lab 9 Blue white selection (screen)

8. Lab 10 and 11 Small scale DNA isolation (Lab 2) Restriction enzyme digestion (Lab 3)

9. Experiment 4 Goal: to characterize the response of light regulated genes. Method: RT-PCR. General take home messages: Use of RT-PCR to measure levels of transcript accumulation; use of mutants to characterize a biological process; environmental regulation of gene expression.

10. Etiolation and det1

11. Experimental set up

12. 1. wild-type grown in the light 2. wild-type grown in the dark 3. det1 grown in the light 4. det1 grown in the dark What are the expression levels of light regulated genes (chlorophyll a/b binding proteins CHAB and CLAB) using tubulin as loading control. -Extract RNA -RT mRNA -PCR cDNA WT-light WT-dark det1-light det1-dark Ladder WT-light WT-dark det1-light det1-dark CHABCLAB TUB CHAB or CLAB Picture of Plants (WT and mutant)

13. Isolation of RNA using Commercial Trizol Reagent Liquid N 2 Trizol Homogenize Phenol Chloroform RNA Isopropyl alcohol Ppt RNA DEPC H 2 O Trizol:-mono-phasic solution of phenol and guanidine isothiocyanate -lyses cells and dissolves cell components while maintaining integrity of RNA Chloroform: separate aqueous and organic phases (RNA is in the top aqueous phase) Isopropanol: Precipitates RNA 75% Ethanol: Wash RNA pellet DEPC (Diethylpyrocarbonate) water: Resuspend RNA pellet in RNAse-free water DEPC interacts with the active site histidine residue of RNAse and irreversibly inactivates RNAses. Aq Org 75% EtOH

14. Assignment 1

15. Example sequence GGTAANGGAACTGGAATCCAAACTCTCTGAAGCTGAGAAGGAATTCATCGA AGGAGCACCAACACGTAGCAAACGATCACCATCCGAGTGGATACCAAGGCCACCCGAAAAATACAGTCTTACTGGGCACA GAGCTCCTATCAACAGAGTTATTTTCCATCCGGTCTTTAGTCTTATAGTATCTGCCAGCGAAGATGCCACTATCAAGGTG TGGGACTTCGAGAGCGGCGAATTCGAAAGAACGTTGAAGGGGCACACCGACAGCGTGCAGGACGTTTCCTTCGACGTCTC CGGGAAACTGTTAGTCTCATGCAGTGCGGACATGTCTATTAAGTTATGGGACTTTCACCAGTCATTCGCCTGCGTGAAAA CCATGCACGGACATGATCACAGTGTCAGCTCTGTCGCATTTGTGCCACAAGGGGATTTCGTAGTGAGCGCCTCTAGGGAT AAGACCATCAAAATATGGGAAGTAGCGACAGGGTATTGTGTCAAAACGTTAACGGGGCACAGAGAATGGGTACGGATGGC CAGAGTCAGTCCTTGTGGAGAATTAATAGCTAGTTGCTCGAACGATCAAACAGTACGGGTTTGGCACGTGGCAACAAAGG AAACGAAGGTCGAACTCAGAGACCACGAACACGTAGTGGAGTGTATCGCATGGGCACCGGACAGTGCAAGAGCATCGATC AACGCTGCTGCAGGGGCGGACAATAAGGGAGCCCATGAAGGACCTTTCCTCGCATCTGGCTCGCGAGACAAAGTAATTCG TGTATGGGATGTCGGTGCCGGTGTTTGTCTCTTCGCCCTATTGGGCCACGACAACTGGGTTCGCGGCATCGTCTTCCATC CTGGTGGCAAGTTCATCGTCAGTGNCTCTGACGACAAGANCCTGCGAGTATNGGANACGCGCAACANANGGGTAATGAAA ACCCTCNAAGCGCACGTCCACTTCTGCNCCTCCNTTGATTTCACAAAAGCCATCCTTACGTGGTCNCCGGTAGTG

16. Step into liquid

17. I will be available for a large-scale public consultation on Friday from 4:00PM until we are done. Bring your laptops. You can make appointments for consultation as well. Time is running out. Assignment 1 due next Monday Oct. 27, 2008 Assignment 1

18. Discussion of Article 3

22. Taken from Hall J. C. Science

23. Taken from Hall Science